Progesterone (4-Pregnene-3,20-dione, hereafter: P4) receptor membrane component (PGRMC) 1 (PGRMC1) is the archetypal member of a divergent eukaryotic branch of the cytochrome b${}_5$ (cytb5) domain supergroup, called the membrane-associated P4 receptor (MAPR) family [1]. The author believes that understanding all PGRMC biology is necessary to provide a conceptual appreciation of the phenomenological interrelationships and implications for its underlying disease-relevant cell biology. Therefore, in perhaps the most extreme manner conceivable, this paper deliberately transgresses the boundaries of looking at strictly disease biology because the most useful perspective for PGRMC is much higher.

We can consider cell and organismal biology at the level of a complex biological program, analogous to a computer program. The many stranded and even root level functionality of PGRMC proteins seems to affect the programmed response at many levels, from cellular to organismal, which in part reflects the evolutionary history of functional acquisition by PGRMC proteins. This constellation is permeated with still intangible potentialities for pharmaceutical intervention in disease processes (such as fertility, metabolic control, cancer, or neurological disorders), and perhaps modulation to extend healthy life. A conceptual understanding of the phenomenological interrelationships between PGRMC functions may be necessary to begin to target specific modularities, while leaving other perhaps critical functions intact. In that respect, it is necessary to first identify all PGRMC functionalities and their interrelationships, before being able to functionally stratify and finally safely pharmacologically address individual functions. To the extent that this is not yet possible, this work is conceptualized as a signpost.

This review attempts to orient the direction of future research by considering the overarching biological landscape, with the conviction that all PGRMC biology is potentially disease-relevant. However, much of the matter covered will be foreign to the medical researcher. Fundamentally, this work pursues the metabolism aspect of PRMC1, including consideration of age-related diseases such as cancer and diabetes, with special focus on the related cell and metabolic changes associated with the neuropathology of Alzheimer’s disease (AD) and possibly aging itself. These will be addressed in the accompanying paper [2] after this present paper builds a conceptual platform.

All cytb5 domain proteins contain a small heme-binding protein fold. The classical mammalian cytb5 proteins, of which humans have separate mitochondrial and endoplasmic reticulum (ER) species, are best characterized for contributing electrons to cytochrome P450 (CYP450) reactions, particularly in the process of steroidogenesis, although that is not their sole function [3]. The MAPR class of non-classical cytb5 proteins were originally identified as having a variably long peptide insertion between helices 3 and 4 of the canonical cytb5 domain fold [1], which forms a loop on the protein surface [4, 5] that I have dubbed the MAPR interhelical insertion region (MIHIR) [6, 7].

It is possible, but not established, that a MAPR protein was present in the last eukaryotic common ancestor (LECA) [8]. This paper will build an argument below that the MIHIRs of possibly the LECA MAPR protein [8], and of the PGRMC protein in the last eumetazoan common ancestor (LEUMCA) [7], were critically important for the origin of eukaryotes and eumetazoans, respectively. Eumetazoans (‘true animals’) are the group of animals including cnidarians (sea anemones, corals, sea pens, jellyfish, box jellies and Hydrozoa, such as Hydra) and bilaterally symmetrical animals.

Because the proposed PGRMC critical functions underpin many foundational aspects of modern mammalian biology, they are proposed to play substantive roles in human disease. This work represents a summary of current knowledge as well as conjectured hypothetical interpretations/interpolations. The author has attempted to make the boundaries between review and conjecture clear. Due to the large scope of the material covered, not all original work in the field has been directly cited, for which the author apologizes to omitted authors. Rather, reviews are cited in many instances. Please see original cited works in those reviews for underlying data.

Animals possess three types of MAPR proteins: PGRMC, Neudesin (NENF) and Neuferricin (NEUFC) [9, 10, 11]. All three families were already represented in the common ancestor of opisthokonts, the eukaryotic group that contains fungi and animals [7]. Humans possess one NENF, one NEUFC and two PGRMC proteins (Fig. 1A, Ref. [6, 7]). PGRMC1 and PGRMC2 arose by presumed gene duplication of an original pgrmc gene prior to the evolution of jawed fish [6, 12]. This paper will not focus on NENF or NEUFC proteins (about which relatively little is known [7]). Rather, it deals primarily with evolutionary events that occurred before the origin of chordates, and therefore prior to the divergence of PGRMC1 and PGRMC2. Accordingly, the paper will refer to PGRMC proteins unless specifically meaning either of vertebrate PGRMC1 or PGRMC2. Unless otherwise stated, any PGRMC amino acid numbering refers to the cognate human PGRMC1 positions.

It can be expected that the two structurally similar PGRMC1 and PGRMC2 proteins exhibit potential functional overlap, but also specific differences (Fig. 1B). Although an analysis of the relationship between PGRMC1 and PGRMC2 merits attention, I will not attempt to address that here. Rather, I will focus on the proposed role of an ancestral MAPR protein in eukaryogenesis, and much later of the proposed involvement of PGRMC in the origins of the gastrulation organizer, and the system of eumetazoan body pattern formation that it may have foundationally enabled. The accompanying paper [2] will consider how this combined biological panoply is relevant for our mechanistic understanding of several modern clinical pathologies.

PGRMC1 was cloned in a variety of different functional contexts in different organisms, including as part of a membrane P4 receptor protein complex, as a dioxin-inducible neural gene, as a protein involved in adrenal steroidogenesis, and as a ventral midline antigen involved in axon guidance during embryological genesis of the central nerve cord. It also emerged that the P4 receptor function of PGRMC1 was important in the female reproductive system, which included expression in both oocytes and their granulosa feeder cells (please refer to original literature cited in previous 2007 reviews [5, 13]).

Granulosa cells were found to exhibit a P4-dependent protection against apoptosis [14]. This P4-induced vitality-conferring function of PGRMC1 was later shown to protect against P4 withdrawal-induced cell death in both granulosa and luteal cells [15] and in MES-SA uterine sarcoma cell [16]. PGRMC1 was later shown to simultaneously decrease proliferation and increase resistance to doxorubicin-induced cell death in a P4-dependent manner in Ishikawa endometrial cancer cells [17], MDA-MB-231 breast cancer cells [18] and MIA PaCa-2 pancreatic cancer cells [19]. This affect appears to be mediated by PGRMC1-dependent CYP450 modification of doxorubicin [4].

When PGRMC1 was discovered to be a CYP450-interacting cytb5 domain protein, it was categorized by annotation data bases as an ER protein and was probably not of much interest to pharmaceutical companies in the early 2000s. However, PGRMC proteins are not your average cytb5 proteins. Tyrosinate heme chelation is unique to the MAPR family and a few bacterial sequences (to be discussed later) among cytb5 proteins [8]. It leads to a characteristically lower redox potential of the heme, and lower affinity for ferrous/reduced heme in the yeast PGRMC-related Dap1 protein [86], in PGRMC1 (K${}_d$ of 2 $\mu$M for ferrous vs 400 pM for ferric) [87], and in PGRMC2 (5.3 $\mu$M for ferrous vs 1.4 $\mu$M for ferric) [85]. In other words, PGRMC1- [85] or PGRMC2- [85] bound heme can dissociate to bind other hemo-proteins after accepting an electron (Fig. 6A, Ref. [4, 8, 86]). Therefore, the redox behavior of tyrosinate heme-chelating MAPR proteins is fundamentally unlike the constant heme occupancy of bis-histidine axially ligated heme [88] found in conventional bacterial and classical eukaryotic cytb5 proteins [8].

The ‘one-hit chemistry’ that can be deductively associated with reduction and disassociation of heme after a PGRMC/CYP450-mediated reaction should lead to the presence of separate heme-bound PGRMC and heme-free apo-PGRMC, which could have dramatically different suites of functions (Fig. 6A), and which has received scant attention. The jettisoned heme must also be transferred to another as-yet unknown protein, which will change its properties. Note once more the argument of McGuire et al. [32] that tyrosinate-chelated heme iron is unlikely to act as an electron carrier.

The level of in vivo PGRMC1 heme occupancy was determined to approximate 0.6 [89], suggesting that populations of holo-PGRMC1 (with heme) and apo-PGRMC1 (without heme) exist. Lee et al. [51] recently argued for a discrete function of “PGRMC1 monomer” involved in glucose regulation after treatment with the small molecule AG-205, which should displace heme. McGuire et al. [32] also identified PGRMC1:cyP450 interactions and cyP450 stabilizations that were independent of heme binding. As will be developed below, the existence of heme-dependent and -independent functions may lie at the crux of both PGRMC1 and eukaryotic biology, with momentous implications for human disease. Lee et al. [51] do not define what is meant by “PGRMC1 monomers”. Nor do they acknowledge that AG-205 effects may be PGRMC1-independent, as explored in the next section.

The effects of PGRMC1 on mitochondria, and its involvement in one of the most conserved eukaryote-specific reactions, prompted the author to explore a possible role in eukaryotic origins. We have recently discovered that MAPR proteins are related to a newly discovered subclass of prokaryotic cytb5 proteins, which we called cytb5M [8, 33]. These proteins that share structural features with MAPR proteins are found in diverse archaea and bacteria and are obviously descended from a very ancient ancestral gene. Hitherto described non-MAPR cytb5 domain proteins chelate heme via a two histidine (2-his) mechanism [86, 103]. Most cytb5M sequences indicated a 2-his mechanism by sequence alignment, and we confirmed this mechanism by solving the crystal structure for two cytb5M proteins [8, 33], albeit with altered orientation of heme binding relative to conventional cytb5 proteins. However, we also discovered a small subset of cytb5M-related proteins which shared with MAPR the tyrosines involved in the otherwise unique tyrosinate heme chelation of MAPR proteins (Fig. 6B,C). We designated this subclass of tyrosinate chelating proteins as cytb5MY (cytb5M with Y heme chelation) [8].

Strikingly, all cytb5MY proteins belonged to the candidate phyla radiation (CPR) bacteria [8] (Fig. 6C), a group of bacteria whose existence only relatively recently became apparent in the modern era of high throughput environmental sequencing. Although no living organism has ever been cultured, they are known from contiguous genome sequences discovered by high throughput environmental sequencing projects, and form a previously unappreciated major branch of life [104, 105]. CPR bacteria are typically obligate symbionts, characterized by reduced genome size, and often lacking genes required for usually essential pathways, such as (and possibly quite relevantly to our story) fatty acid synthesis [106, 107, 108].

It remains unclear how the membrane lipids of CPR are derived, since they cannot de novo synthesize them, and all biological membranes are synthesized in pre-existing membranes. This may be relevant for eukaryogenesis, as argued below, if a CPR lipid transfer system involving a cytb5MY protein was co-opted by the proto-eukaryote. It therefore appears quite possible that MAPR proteins entered the genome of the last eukaryotic common ancestor (LECA) from a symbiotic CPR bacterial genome. However, it remains possible that cytb5MY proteins are the result of horizontal gene transfer from a eukaryote with subsequent loss of the MIHIR region. Despite trying very hard, we could not resolve this issue [8]. The following discussion considers the implications if the former is the case, and CPR cytb5MY proteins were involved in eukaryogenesis.

Analysis of CPR genomes identified a consensus operon structure, which may have resembled the one involved in a putative eukaryogenic CPR bacterium (Fig. 6D). Known CPR cytb5MY genes are often found in operons containing cytb5MY, two other cytb5-related proteins (with 2-his heme chelation), a putative ferric reductase-like (pFre) gene, another gene of unknown function, a two-component inducible signal transduction unit, and possibly a gene for heme- and sterol-binding tryptophan-rich sensory protein (TSPO), also known as translocator protein, or peripheral benzodiazepine receptor (PBR). All proteins are transmembrane proteins. Since bacterial operons often encode proteins of related function, these genes in extant CPR genomes probably encode proteins that contribute to an inducible redox-related membrane complex [8]. It is tempting to speculate that the pFre enzyme alters cytb5MY heme iron oxidation to accept or jettison heme (Fig. 6A), however that remains speculative.

Nothing more is currently known about CPR cytb5MY proteins. However, we can reasonably speculate about at least one role played by cytb5MY if CPR were involved in the eukaryogenic symbiosis. From yeasts to mammals [31, 68] PGRMC-like proteins interact with CYP51A1 to demethylate lanosterol (Fig. 7A, Ref. [32]). CYP51A1 is the most strongly conserved eukaryotic cyP450 enzyme [39], suggesting that it serves a centrally important role in eukaryotic biology.

Lanosterol, the first steroid produced, is a five-ring organic molecule synthesized as a product of the mevalonate pathway (MVP). The triterpenoid squalene is formed by the condensation of six isoprene units by the MVP and cyclized into lanosterol involving the combined action of first squalene epoxidase and then oxidosqualene cyclase (also called lanosterol synthase), which produce a characteristic hydroxyl on the 3 position of sterols (Fig. 2). MVPs can be found in organisms from all kingdoms of life. In bacteria, squalene is also produced via the MVP and cyclized to six-ringed structures called hopanoids by squalene-hopene cyclase (SHC) enzymes that share distant homology with eukaryotic squalene cyclases [34, 109]. Lanosterol can be therefore regarded as an atypical 3-hydroxylated eukaryotic hopanoid that is largely produced by originally bacterially descended genes. The bacterium that most critically required regulation in our eukaryotic family tree was the proto-mitochondrion.

The synthesis of cholesterol from lanosterol produces diverse intermediary bioactive sterols (Fig. 3), which can each evoke their own biological responses fully independently of cholesterol production [36, 110]. Obviously, the very first of these reactions evolved first. It is the one involving lanosterol 14-demethylation catalyzed by PGRMC1 and CYP51A1 (Figs. 2,3). Our analysis showed that the required enzymes, including the genes for the MVP, squalene cyclase (which were previously known), CYP51A1 and PGRMC, all originated in bacterial rather than archaeal genomes [8]. Therefore, it is probable that a lanosterol-like hopanoid was demethylated by a cytb5MY/CYP51A1-like reaction in the symbiotic proto-eukaryote during the protoeukaryotic evolutionary response to molecular oxygen. (This has the status of reasonable speculation).

Interestingly in this context, oxygen levels regulate both the proteolytic activation, stability, and subsequent proteasomal degradation of the Sre1 homolog to mammalian SREBP proteins (which induce mevalonate pathway and steroidogenesis), whereby the MVP is upregulated under hypoxic conditions to compensate for lowered levels of oxygen-dependent steroid synthesis. Sre1 is destabilized in the presence of oxygen when sufficient steroids are available. Sterol synthesis in this system is oxygen-dependent, and also inhibits Sre1 proteolytic activation [111, 112]. Therefore, in yeast, steroidogenesis is regulated by oxygen, and occurs in the presence of oxygen.

It has long been noted that the squalene monooxygenase and CYP51A/PGRMC reactions at the base of eukaryotic sterol synthesis both require molecular oxygen (Fig. 2), which could mean that they first appeared as a response to elevated levels of oxygen. However, 3-OH sterols appear in the fossil record about a billion years before eukaryotes are thought to have arisen, and alternative anaerobic reactions (e.g., $\beta$-oxidation hydroxylation-type addition of water across double bonds) could conceivably have enzymatically produced sterols prior to the post-photosynthetic global rise in oxygen levels [35]. The restriction of sterols to eukaryotes and the current dating of eukaryogenesis as a post-photosynthetic event (see the next section below) make a strong if not compelling argument that the evolution of modern eukaryotic steroidogenesis was somehow associated with eukaryogenesis. Earlier anaerobically-produced sterols [35] may have been produced by a lineage, one of whose members could have contributed to eukaryogenesis, and is perhaps now extinct.

Notwithstanding, and conceptually noted here for the first time, the combination of tyrosinate heme chelation and dissociation leading to altered MAPR states, as well as the dependence of the squalene monooxygenase and cytb5MY/CYP51A1 reactions on oxygen, potentially enabled a regulated response to oxygen by early eukaryotes (Fig. 2) (and the regulation of yeast SREBP pathway leading to MVP induction by oxygen levels [68, 112]). This would provide a potential mechanism to regulate the metabolic activity of the proto-mitochondrion. In this respect, it is striking that PGRMC2 regulates mitochondrial gene expression by reversible binding and shuttling of heme from the mitochondria (where heme is synthesized) to the nucleus (where genes encoding most mitochondrial proteins are located) [82] and PGRMC1 can regulate mitochondrial association with the ER [113].

Hopanoids lower the permeability of bacterial membranes to protons, which increases the efficiency of electron transport chains by increasing the proton gradient [34, 109]. Mitochondrial membranes require cholesterol to function, although elevated levels can be pathological [114]. A model emerges where genes from a symbiotic CPR bacterium were potentially involved in regulating the metabolic activity of the proto-mitochondrion in a symbiotic proto-eukaryotic cell collective by regulating sterol synthesis and transport (Fig. 7). This model is hypothetical and requires substantiation.

Because many CPR are unable to synthesize fatty acids, they must be able to somehow ferry membranes or at least lipids from host cell membranes. In this respect, the membrane trafficking role of MAPR proteins may have been involved in the eukaryotic origins of vesicle trafficking, or of lipid droplet formation. This model is even more conjectural and is also unsubstantiated.

This all implicates MAPR proteins with a previously unimagined (and still uncertain) role in the origins of eukaryotes. Strikingly, the products of the PGRMC1/CYP51A1 reaction in mammals are either follicular fluid meiosis-activating sterol (FF-MAS) or dihydro-FF-MAS (Figs. 2,3) [36, 110], which are both inducers of meiosis, a major innovation of the LECA relative to its prokaryotic forebears [115]. The association of PGRMC1 with the mitotic spindle kinetochore [57, 61] and its role in ensuring the ‘faithful progression through mitosis and meiosis’ [57] are therefore most interesting in this context (See also the review by Lodde et al. [116]). To better develop this theme and garner an appreciation for the importance of oxygen to eukaryotic origins, we should consider eukaryogenesis in closer detail. We (eukaryotes) are children of oxygen who would not have evolved in a world without photosynthesis.

In terms of this review, we can view autophagy as a system for generating non-glucose carbon skeletons for metabolic energy production, and hence we can place autophagy near the metabolic functional heuristic core of PGRMC1’s proposed ancestral eukaryogenic role (regulating energy production by mitochondria in response to oxygen levels). Autophagy plays a critical role in the metabolism and clinical phenotype of many tumors [135, 136], and other pathologies such as Alzheimer’s disease [137]. PGRMC1 regulates the induction autophagy by forming protein complexes with MAP1LC3 (microtubule-associated protein 1 light chain 3, or LC3) and UVRAG (UV radiation resistance associated/UV radiation associated gene) [138].

PGRMC1-dependent autophagy can be pharmacologically activated to metabolically render cultured ovarian cancer cells more likely to undergo apoptosis, and more sensitive to cis-platin treatment [139]. PGRMC1 knockdown promotes the differentiation of human pluripotential stem cells coincidentally with a reduction in autophagy and elevated activity of the p53 and Wnt/$\beta$-catenin pathways, which leads to loss of pluripotency and the differentiation into multiple cell types [140], consistent with a central role of autophagy and metabolism in maintenance of the autonomous single celled/stem cell state, which may reflect metabolic changes which permitted the evolution of the gastrulation organizer and therefore the LEUMCA. Note that p53 is also a heme-dependent protein [80, 81], which could therefore also be targeted by PGRMC nuclear heme shuttling [82] independently of Wnt signaling.

Organizer activity requires Wnt/$\beta$-catenin signaling. PGRMC1 Sumoylation is associated with a nuclear subcellular localization [49, 141, 142]. There, it associates with promoters with binding sites for the TCF/Lef transcription factor, where Tcf/Lef-dependent transcriptional activity is enhanced in the absence of P4, or suppressed in the presence of P4 [142, 143, 144]. TCF/Lef is the target of the Wnt/$\beta$-catenin pathway, which will be discussed further in the accompanying manuscript [2]. TCF/Lef target promoters include many immediate early genes, including c-myc, whose induction leads to increased metabolic activity in the G1 cell cycle phase, and promotes regulatable transition of the G1 checkpoint and entry to the cell cycle, at least in granulosa cells [144]. PGRMC1 and PGRMC2 control a G1 checkpoint mechanism, where depletion of either increases entry to S-Phase, but does not result in cell proliferation [56, 59]. This is presumably because PGRMC1 associates with kinetochore microtubules and the chromosomal kinetochore during mitosis and meiosis [57, 60, 61, 116, 145]. The ability of P4 to suppress transition through the G1 checkpoint involves a cytoplasmic complex between PGRMC1/PGRMC2 and PAQR7 [59]. The latter is a P4-receptor of the of the G-coupled serpentine class II progestin and adipoQ receptor (PAQR) proteins, which are also called membrane progestin receptors (mPRs) and include: mPR$\alpha$ (PAQR7), mPR$\beta$ (PAQR8), mPR$\gamma$ (PAQR5), mPR$\delta$ (PAQR6), and mPR$\epsilon$ (PAQR9) [21, 146]. PAQR/mPR protein functions have been recently reviewed [147].

It has been proposed that (at least part of) PGRMC1’s P4-responsiveness may be ascribable to interactions with PAQR proteins [20, 47]. With respect to eukaryotic origins, it is notable that whereas most G-coupled protein receptors originated from the archaeal host cell, the PAQR-type proteins are of bacterial origin [148]. It is therefore conceivable that a MAPR/PAQR-GPCR response was involved in metabolic regulation of proto-mitochondrial metabolism. In this context it is also noteworthy that the PGRMC1-dependent P4 induction of glycolytic Warburg metabolism in HEK293 cells involves G$\alpha$ proteins and $\beta$-arrestins, which are required for PGRMC1 degradation and attenuation of glycolysis after P4 treatment [52]. Involvement of $\beta$-arrestins in PGRMC biology is of great interest because, like PGRMC1 [149, 150], $\beta$-arrestin-2 enhances endocytosis of the LDLR [151]. Arrestins also form scaffolds which link mitogen-activated protein kinase (MAPK) signaling to GPCR activity [152].

That such immediate-early gene induction of anabolic metabolic phenomena by PGRMC1 may be widespread is consistent with observations e.g., from neural progenitor cells that P4 increases cell cycle gene expression and proliferation in a PGRMC1/2-dependent manner [58]. Therefore, autophagy may be inversely related to active mitogenic signaling, suppressing Wnt signaling while the cell hunkers down to inhibit cell division until environmental nutrient scarcity is overcome. Direct regulatory modulation of the Wnt and autophagy pathways by PGRMC1 is consistent with the ancient eukaryotic role of PGRMC1 proposed in this work and may be related to the metabolic control of the Warburg Effect.

A MAPR-regulated control of metabolism via the autophagic supply of amino acid fuel supply may be ancient in eukaryotes, since the plant MAPR protein MSBP1 (Membrane Steroid Binding Protein 1) associated with starvation-induced components of the reticulophagy (the selective autophagy of ER proteins) system [153]. The MAPR protein was identified as a substrate for autophagy in this plant system. It would be interesting to assay whether reticulophagy would operate without MSBP1, if indeed autophagy is part of an ancient MAPR-dependent mechanism of metabolic regulation related to eukaryogenesis.

A main theme of this present paper, extending thoughts from a previous publication [8], is to consider the implications if the ancestral CPR ctyb5MY protein that gave rise to MAPR proteins like PGRMC was indeed involved in the regulation of mitochondrial activity and cell metabolism in the LECA (which may or may not be true). In any case, a relatively early eukaryote must have acquired a MAPR gene. It may have come from an inducible CPR operon (which contained a two-component element) that contained a putative ferric reductase and three cytb5-domain proteins: the cytb5MY gene and two others which are unrelated to eukaryotic genes (Fig. 6D). Only MAPR proteins are identifiably related among eukaryotic proteins to this consensus CPR operon. However, the operon in several CPR species also contains the TSPO gene [8], suggesting possible functional association between MAPR proteins and TSPO (Whether eukaryotic TSPO is descended from CPR bacteria was not examined in detail, but CPR TSPO proteins do not give the highest BLAST scores compared to other bacteria using human TSPO as search query, and therefore CPR TSPO is unlikely to have been ancestral to the eukaryotic proteins).

TSPO proteins are highly evolutionarily conserved, being found in eukaryotes, bacteria and archaea [154, 155]. Like PGRMC1, mammalian TSPO’s functions remain elusive. It is observed at several different subcellular locations: primarily the mitochondrial outer membrane, but also the cytoplasmic membrane as well as nuclear and perinuclear localization following stresses such as injury or inflammation [156, 157]. Its natural ligands are suggested to include heme, cholesterol, and the protein called Diazepam Binding Inhibitor (DBI) (also known as Acyl-CoA-binding protein), an acyl-CoA ester-binding protein [158]. However, the physiological relevance of identified ligands remains unclear [155]. TSPO plays prominent roles in oxidative stress response and inflammation and has been strongly associated with steroidogenic tissues (for reviews, see [154, 158]).

Earlier literature associated TSPO with steroidogenesis and cholesterol transport into the mitochondria, however it has now become clear that despite high expression in steroidogenic cells, TSPO is not required for steroidogenesis [159]. It does appear linked to mitochondrial fatty acid oxidation [160]. Hiser et al. [154] propose that TSPO was originally a porphyrin-binding bacterial stress protein, which has gained new functions in the course of evolution. Its contributions to redox and inflammatory homeostasis [154] are consistent with that conclusion. If an original steroidogenic cytb5MY protein was co-expressed in a CPR operon with a TSPO allele [8], then TSPO and PGRMC1 may jointly or reciprocally modulate some ancient eukaryotic processes.

Lee et al. [161] show that PGRMC1 is involved in a pro-inflammatory response involving EGFR in hepatocellular carcinoma. It will be interesting to see whether TSPO is involved in that system. Considering the involvement of PGRMC1 in regulating fatty acid metabolism [41, 43, 162, 163, 164, 165], and PGRMC1’s profound effects on mitochondria [50, 113, 165], and its association with stress response [96, 97, 98, 99], the overlap between TSPO and PGRMC1 biology is extensive, and fully consistent with an ancestral relationship. It is tempting to speculate that a cytb5MY protein might have transferred its heme to TSPO in a redox-dependent manner, thereby changing the responsiveness of cytb5MY and TSPO.

In preliminary work we show that TSPO is required for sigma-2 receptor (S2R) activity of TMEM97 in at least MIA PaCa-2 pancreatic cancer cell binding of the fluorescent SW-120 S2R ligand [166]. TMEM97 and PGRMC1 form a protein complex that is involved in membrane trafficking [149, 150, 167], and like PGRMC1, TMEM97 is implicated in sterol biology (for review: [168]). Recently 20(S)-hydroxycholesterol has been shown to be an endogenous ligand of TMEM97 [45]. We did not observe interaction between PGRMC1 and TSPO, however, both TSPO and PGRMC1 interacted with TMEM97 by proximity ligation assay [166]. Those preliminary results are currently being investigated further. The CPR operon structure is clearly involved in some inducible redox process, and the cytb5MY gene would have been of such central importance to eukaryogenesis that it could not be lost to eukaryotes.

One of the common features of eukaryotic cells from that time to this is the mitochondrion. PGRMC has coevolved with several classes of mitochondrial genes [67], and PGRMC1 phosphorylation status imposes quite dramatic changes on the way mitochondria behave [50], as if a spanner had been thrown into the mechanism of this ancient system. As intriguing and plausible as this hypothesis is, it clearly requires further substantiating validation.

The requirement of the PGRMC/CYP51A1 most highly conserved eukaryotic cyP450 reaction for molecular oxygen (Fig. 7A) is extremely relevant in this context. It allows us to develop a hypothetical scenario where in the presence of oxygen, lanosterol is demethylated to produce follicular fluid meiosis-activating sterol (FF-MAS). The resulting reduced heme would dissociate from PGRMC, potentially creating a binding site for the newly formed FF-MAS (Fig. 7B). This conceivably also facilitates a newly acquired eukaryotic cytb5MY membrane trafficking function via the MIHIR, which is not present in known CPR cytb5MY genes but was acquired by the first eukaryotic MAPR gene (whether or not CPR were involved) (Fig. 6B,C). This is all speculative.

Interestingly most CPR cytb5MY contain two glycine residues at the point where the MIHIR is inserted (Fig. 6C). The GG sequence would enable a sharp turn in the polypeptide backbone between the two helices present in the canonical cytb5 fold [8]. This corresponds in the sequence alignment of Fig. 6C to PGRMC1 C129/L130 which mediate direct hydrophobic contacts with heme [4]. Insertion of the MIHIR sequence between these two helices, at a point of heme contact (Fig. 6C), was the historical original defining feature of the MAPR family [1], in which those two glycines are not conserved. It would be surprising if MIHIR function and heme occupancy were not interrelated.

The proto-MAPR protein may have transported a sterol/hopanoid to the mitochondrial membrane to activate the TCA cycle in the presence of oxygen (Fig. 7). The sterol-associated biology involved still seems to be mirrored in modern PGRMC1 biology [47, 67, 150]. Heme chaperoning or regulation of heme synthesis [67, 82, 85, 169] may also have been involved. This, of course, is hypothetical speculation.

Cytoplasmic membrane trafficking itself, where vesicles are pinched into the cytoplasm in an actin-dependent process, was thought to have arisen newly in eukaryotes [170], however genes for both a dynamic actin cytoskeleton and cytoplasmic vesicle transport are widespread throughout the Asgard archaea [128]. Motivated from this perspective, we recently identified that the PGRMC1 MIHIR contains a predicted coiled-coil protein interaction motif that is like motifs in many myosin proteins [7]. A coiled coil is a protein interaction motif where alpha-helices from two protein interact with each other via hydrophobic and other interactions between two helices. Because of the helical register of residues, aliphatic residues are spaced with a heptad repeat pattern in the sequence [171].

Myosins are the motor proteins associated with the actin cytoskeleton, and the implication is that PGRMC1 interacts with some of the same proteins as myosins. We [91] and others [172] have discovered that PGRMC1 is present in protein complexes with mitochondrial proteins, and with components of the actin cytoskeleton. The latter interactions are sensitive to the small molecule inhibitor AG-205 [91], which was designed to occupy the MAPR heme-binding site [173] (But see Section 2.1.1 above.)

Considering the above, the new eukaryotic acquisition of the MAPR MIHIR was conceivably related to eukaryogenesis: perhaps originally involving CPR membrane exchange because many CPR bacteria cannot synthesize their own fatty acids, and the MIHIR was probably required in the LECA. The hypothesis here is that MAPR membrane trafficking was involved in supplying MAPR-synthesized sterols and/or perhaps heme to regulate the mitochondrion in the presence of oxygen. It has been observed that eukaryogenesis must have been an inherently improbable event, having arisen in this solar system with the same frequency as life itself (once each) [170]. Perhaps the unique cytb5MY with a membrane-trafficking MIHIR was instrumentally enabling in the process. Clearly, this field merits further research.

Hydrophobic heptad repeat residues of the predicted coiled-coil region of the MIHIR (Fig. 8, Ref. [7, 53, 174, 175, 176, 177, 178, 179]) are conserved in MAPR proteins (although predicted coiled-coil formation is not conserved) [7], and appear to be inherited from the ancestral eukaryotic MAPR MIHIR sequence. This was inserted into the MAPR cytb5 domain fold in the middle of the heme-binding domain. Heme interacting Y107 (H-bond) and Y113 (tyrosinate heme chelation), and multiple hydrophobic interactions with heme (G124, L125, F128, C129 and L130) are N-terminal to the MIHIR, whereas propionate H-bond forming K163 and Y164 are C-terminal to the MIHIR (Fig. 6C) [4, 180]. We can then imagine that the MIHIR, or proteins interacting with it, could exert strong allosteric influence over heme affinity, and probably vice-versa. This interrelationship is here hypothesized to have been critical in eukaryogenesis, a process which involved the host cell and mitochondria adapting to each other permanently.

Peluso and Pru [181] report that PGRMC1 associates with eukaryotic ribosomal translation initiation factors. In a pilot co-IP and proteomic protein identification experiment, Sarah Teakel in the author’s lab had also identified ribosomal proteins in the co-IP pellets using HA-tagged PGRMC1 as bait. For reasons explained elsewhere, due to lack of resources we were unable to generate replicate numbers to provide data of publication quality, and so were unable to pursue this. We also already had the proteomics pathways analysis that was later published in 2020 [50], suggesting that PGRMC1 phosphorylation status could affect the abundance of some proteins involved in translation, but also that the dramatic changes in protein abundance profiles that we observed could be due to alterations in the subset of cellular mRNAs being translated. That prompted the author to investigate hints of ribosomal association from published resources, where The Human Protein Atlas reported prominent localization of PGRMC1 to the nucleolus, the site of ribosome biogenesis, and speculate that PGRMC1 “may affect the composition of ribosomes, translation initiation factors” [47], as cited by Peluso and Pru [181]. Terzaghi et al. [182] later reported the nucleolar localization (i.e., the site of ribosomal RNA synthesis and pre-ribosomal particle assembly [183]) of PGRMC1 in bovine granulosa cells and oocytes.

We later discovered that PGRMC1 was inducing dramatic epigenetic changes in the MIA PaCa-2 cells we worked on, which could have been sufficient to induce the observed large changes in protein expression without the need to invoke effects on ribosomes and selective mRNA translation. However, the possibility that PGRMC1 can affect protein expression at the translational level gains credence from the report of Peluso and Pru. Like our lab, they had also been investigating PGRMC1-ribosomal interactions prior to 2016, and they recently reported the presence of several ribosomal elongation initiation factors in PGRMC1and PGRMC1 co-IP pellets [181]. To consider just one example, for PGRMC1 these included the Eukaryotic Initiation Factor 3 (EIF3) subunit EIF3B. The multi-component EIF3 complex binds to and regulates the translational efficiency of specific mRNAs involved in growth control, cell cycle, differentiation, and viability [184]. Its activity can be regulated by methylation via EEF1AKNMT (eEF1A lysine and N-terminal methyltransferase), which can affect the subset of mRNAs being translated and tumorigenicity [185, 186]. Therefore, PGRMC1 could possibly modulate the efficiency with which specific EIF3B-depdendent mRNAs (for example) are translated. This concept extends to other ribosomal factors.

Having spent many years working in the field of proteomics, the author experienced first-hand the developing awareness that mRNA levels are surprisingly poorly correlated with protein levels. It is now an accepted phenomenon that many mRNAs are only translated under appropriate regulated circumstances [187, 188, 189, 190, 191].

This point is expounded here in detail because this biology could well represent vestiges of an ancestral cytb5MY/MAPR role from eukaryogenic times, where modulation of host cell metabolism by an obligate symbiotic CPR bacterium may have been mediated via a cytb5MY/MAPR protein which was present in the host cytoplasm. This hypothesis predicts that PGRMC (and there is no reason to assume that either modern PGRMC1 or PGRMC2 functions are more typical of an ancestral state) modulation of ribosomal function would probably have initially served to alter host cell metabolism to provide metabolites for the obligate CPR symbiote. Such a switch may have controlled feeding carbon skeletons to either oxidative phosphorylation (mitochondria) or cytoplasmic lipid or amino acid syntheses. As such, PGRMC modulation of ribosomal function would be consistent with the hypothetical eukaryogenic role of MAPR proteins proposed in this current work. If this is correct then PGRMC1 modulation of ribosomal activity is predicted to modulate metabolism via regulation of mitochondrial function, and perhaps even cell cycle control if that is inherited from early eukaryotes. Please note that (1) the involvement of PGRMC1 in ribosomal translation remains formally unproven, and (2) even such proof would not lead to automatic acceptance of the eukaryogenic hypothesis. This avenue requires further investigation.

As time passed after the appearance of the LECA, eukaryotes diversified into multiple lineages, one of which gave rise to both fungi and humans. That lineage is called the opisthokonts, of which a subgroup called the holozoans became sophisticated predators able to engulf bacteria and feed off the cytoplasmic contents of eukaryotes as large as themselves [193]. This group developed a toolkit of new genes that would prove essential for subsequent animal evolution, importantly including the evolution of both tyrosine kinases, and SH2 domains that bind to phosphorylated tyrosines, which are absent from fungi but present in animals and their close protozoan relatives such as the Choanoflagellates (the sister group to animals) [73].

Choanoflagellates are the closest single-celled protozoans to animals, and multicellular animals share a common ancestor with that group [73, 194, 195]. Some choanoflagellates can form multicellular spheres, which are topologically like early animal embryos of a single cell layer sphere. There is superficial resemblance of the body organization of sponges or placozoans to multicellular choanoflagellates, featuring a layer of polarized flagellated cells that obtain sustenance from the exterior and achieve complex surface topologies via adherens junction-mediated movements by coordinated action of myosin-motored actin cytoskeleton (actomyosin) and adherens junctions [195]. However, a fundamental difference in body topology is that planar coalescences (sheets) of separate coalesced choanoflagellate cells can actively form a sphere. By contrast, the spherical blastula of all animals arises from the serial divisions of a single zygote cell, which is never observed in choanoflagellates [196].

The first animals evolved from a common ancestor with choanoflagellates sometime between perhaps 950 and 750 million years ago (mya) [197, 198], perhaps close to 800 mya (Fig. 8A), which coincided with the appearance of a suite of new metazoan-specific genes [197]. This was a time of low organic productivity, which was subsequently reduced even further by the approximately 75 million-year Sturtian glaciation event: the most prominent of the Neoproterozoic “Snowball Earth” glaciations thought to have frozen the world’s oceans. See also Gold for further consideration of this topic [175].

The Sturtian glaciation led to dramatically reduced bioproductivity, which corresponded to both reduced organic biomass availability, and even lower oceanic oxygen levels [174]. If the gastrulation organizer arose as a response to variable oxygen conditions, that could be retained in modern early embryological processes. Intriguingly, pluripotential embryonic stem cells (PSCs) exhibit a plastic metabolic state that can reversibly adapt to fluctuations in oxygen concentration. The most conspicuous two states have been described as “naïve” and “primed” PSCs. Naïve cells arise shortly after the zygote begins transcribing its genome, which rapidly becomes hypomethylated to remove parental epigenetic status. This involves net low methylation levels of genomic CpG and the repressive H3K27me3 (trimethylation at lysine 27 of histone H3). Naïve PSCs are found in the inner cell mass of the early blastocyst, which emerges with gastrulation, and can interconvertibly obtain ATP via either anaerobic glycolysis or aerobic oxidative phosphorylation (a metabolic switch that was implicated PGRMC1 with above).

During embryology, naïve cells later develop into primed PSCs, which in the mouse exhibit predominantly glycolytic metabolism, as well as hypermethylation of CpG sites and the repressive combination of histone H3K27me3 and H3K9me2/3 (di-methylation at lysine 9 of histone H3) methylation. Certain enhancer and promoter regions remain hypomethylated. In culture, the naïve-primed conversion is reversible, depending upon culture conditions [199, 200, 201, 202, 203], which include switching from 5% to 20% oxygen [204]. Accordingly, the implantation stage mammalian blastocyst is almost exclusively glycolytic [205, 206]. These are the developmental processes which give rise to the gastrulation organizer, and which probably somehow reflect its evolution to produce the LEUMCA, and perhaps the environmental adaptations required at that time.

If Sperling and Stockey’s estimated chronological calibration of major metazoan lineage divergences is correct, then the LEUMCA common ancestor of bilaterians and cnidarians arose perhaps just over 700 mya, corresponding roughly to the origin of the Sturtian glaciation (Fig. 8A). However, this figure represents the consensus from several different studies, each of which was associated with larger 95% confidence intervals. See the original work for details [174].

It is possible that multicellularity arose with the ancestor of choanoflagellates, or even earlier, and that modern metazoans had multiple choanoflagellate-like origins. However, it is also possible and perhaps more likely that animal multicellularity arose with an “Urmetazoan” common ancestor of poriferans, ctenophores, placozoans, cnidarians and bilaterians [194].

Much of the uncertainty has concerned the position of the ambiguous group known as ctenophores, which have a gut, mesoderm-derived muscle and a nervous system with synapses [207]. Placozoans and poriferans do not, although poriferans do contain many genes related to synapses (as indeed do the choanoflagellate single celled animal sister group) [208]. This is important when considering the origins of complex animal bodies, and especially nerves, in our direct lineage.

Ctenophores have ectodermal and endodermal nerve nets, but lack many synaptic genes present in the LEUMCA clade. It has long been controversial whether nerves evolved separately in LEUMCA and ctenophores. However, ctenophore nerves are specified late in development and related to ectodermal cells, in contrast to cnidarians where cells of the nerve net differentiate early in development from both ectodermal and endodermal cells [207].

Ctenophore early embryonic signaling molecules are also different to those of eumetazoans in terms of expression patterns and chemical identity. Furthermore, many traditional bilaterian neuronal markers are shared with cnidarians but absent from ctenophores. Like cnidarians, ctenophores have tentacles, a gut, nerves, and polarized epithelial cells. Their comparative embryological development, based upon comparison of different genetic pathways in the ctenophores than in cnidarians and bilaterians, provides very strong evidence for independent evolution of nerves (and therefore gut and muscle) in the LEUMCA and ctenophores. The complement of genes in the genetic toolkit of ctenophores is also more like poriferans than any other animal group. For such reasons, many recent authors have concluded that ctenophores are a sister group to all other metazoans [74, 177, 179, 194, 195, 209, 210, 211, 212, 213]. Ctenophores could even represent a separate evolution of multicellularity from a different choanoflagellate-like group than other metazoans [177]. From our own work, based only on MAPR proteins, the NEUFC C-terminus suggests that ctenophores share derived features with poriferans and other animals relative to at least the choanoflagellates we surveyed, while the PGRMC C-terminus supports a common ancestor of placozoans and eumetazoans but not poriferans or ctenophores [7], supporting the tree topology of Fig. 8A (albeit on the basis of just two genes).

Erives and Fritzsch [176] offer compelling evidence, based on shared inheritance of paralogous duplicated genes, that ctenophores indeed do form a sister group to all other metazoans, which has been followed in Fig. 8A. For that reason, ctenophores are not further considered in this paper, which assumes that the first appearance of nerves in the human lineage was in the LEUMCA. However, note that while it seems to be accepted that ctenophores are not descended from the LEUMCA, it remains debated by some whether an ancestral animal may have evolved nerves with subsequent loss by poriferans and placozoans [211].

The major animal multicellularity innovation was achieved prior to the LEUMCA: cadherin-mediated adhesion of cells to form an epithelial layer attached basally to a strong and flexible basal lamina enabled directed cadherin dependent and adherens junction-mediated shape changes by the actin cytoskeleton. This enabled the epithelial layer to undergo morphogenesis from an ancestral spherical shape into convoluted cup or tubular forms, as evidenced by early metazoan body grades best represented today by poriferans and placozoans [195, 214]. Cadherins themselves appeared before metazoans, however metazoan evolution was accompanied by the appearance of metazoan-specific genes with regulatory features on the cytoplasmic tails, as well as a small number of accessory proteins, to interact with them [215].

The LEUMCA developed new actin innovations. The type of muscle possessed by cnidarians permits us to reconstruct the probable LEUMCA situation. Although cnidarians vary considerably between genera, their body consists of two cell layers of primarily epithelial cells, with some ganglion neurons, sensory neurons, gland cells, and other specialized cnidarian cell types including nematocyte and nematoblast cells (involved in toxin delivery). Cnidarian muscle cells are epitheliomuscular, meaning they have both epithelial and contractile character. Perpendicular networks of ring muscle and longitudinal muscle fibers connect the adherens junctions of adjacent epithelial cells, which enables peristaltic motion (e.g., the swimming action of a jellyfish) [216, 217].

The novel cadherins described above led to regulatable adherens junction activity in eumetazoans, adding new function to a pre-existing ancient machinery that connected to the actin cytoskeleton via $\beta$-catenin and vinculin [215] (Fig. 9). Desomosomal cadherins, linking the intermediate filament networks of adjacent cells via desmosomes, are a much later vertebrate innovation [214].

Let us revisit consideration of the MIHIR, which appeared in the first MAPR protein early in eukaryotic evolution as discussed above. Early animal acquisition of Y139 in the MIHIR [7] may have contributed to eumetazoan evolution. It has been speculated but not demonstrated that the MIHIR may be involved in the attested membrane trafficking ability of PGRMC1 (for review: [47]). PGRMC1 is known to interact with components of the actin cytoskeleton [91, 172], and a portion of the MIHIR exhibits both strong evolutionary conservation and predicted propensity to form a coiled-coil protein interaction involving heptad repeat L residues discussed above (Fig. 10, Ref. [7]).

That region is part of a motif which resembles similar motifs from the coiled-coil regions of multiple myosins [7], and would present a conserved negatively charged amphipathic helical surface (Fig. 10C). It is conceivable that an amphipathic helix is somehow involved in PGRMC1’s membrane trafficking function, however reported membrane trafficking proteins with amphipathic helices contain positively charged, or mixed positively and negatively charged helices, where the positive charges interact with negatively charged membrane lipids [218, 219]. It seems therefore more likely that this PGRMC1 putative amphipathic helix interacts with positively charged proteins (such as those involved in membrane trafficking?) that can also interact with selected myosins sharing the similar motif [7]. Thereby PGRMC1 would compete with myosin motor proteins for binding to some proteins (which could also regulate motive force required for membrane trafficking).

Notably, PGRMC1 Y139 would be one of the aliphatic residues that would contribute to coiled-coil inter-helical protein interaction surface (Fig. 9). This residue appears to have been a tryptophan in the single celled ancestors of animals, but to have mutated to a tyrosine by the time of the LEUMCA [7]. Both tryptophan and tyrosine, being large hydropathic residues, should be compatible with a hydrophobic surface involved in coiled-coil formation. However, the conserved aliphatic helical surface of Fig. 9C would be destroyed by phosphorylation of Y139. This would (1) interrupt any coiled-coil interaction, and (2) facilitate new interactions with SH2 domain proteins [7] (Fig. 8B).

Therefore, we can imagine that the LEUMCA could retain whatever functions were performed by the predicted ancestral hydrophobic helical surface, but that it could switch these off by Y139 phosphorylation. Thereby, novel functions associated with the recruitment of SH2 domain proteins to phosphorylated Y139 hypothetically provided new functions and potentially a new platform for eumetazoan evolutionary innovation. This system may have produced the gastrulation organiser of eumetazoans, which formed the foundations upon which subsequent chordate CpG epigenetic gene regulation evolved to regulate tissue-specific gene expression.

While the putative coiled-coil helical MIHIR region is not helical in the PGRMC1 crystal structure [4], the evolutionary conservation of Fig. 9B suggests evolutionary selective pressure to form a helix. If a helix does form, since the MIHIR is situated immediately between two helices which make direct heme contacts [4], it is highly likely that allosteric conformational changes throughout the protein would alter the geometry of heme-interacting residues and reduce affinity for heme. Indeed Y113 (the tyrosinate heme iron chelating residue [4]) is among the most frequently observed phosphorylation sites for PGRMC1 [66], and phosphorylation of Y113 would be incompatible with heme binding. Y113 is also the phosphate acceptor of a conserved YxxL/I immunoreceptor tyrosine-based activation motif (ITAM) [5, 220]. Phosphorylated ITAMs were first associated with membrane trafficking of non-catalytic tyrosine-phosphorylated receptors [221].

Accordingly, it is fully appropriate to think of PGRMC function in terms of heme-dependent and -independent different roles for apo- and holo-PRGRMC. Any association between the MIHIR, membrane trafficking, or motility remain hypothetical and must yet be experimentally explored. However, the membrane trafficking role of PGRMC1 is involved in the pathogenesis caused by synaptic oligomers of A$\beta$o protein in AD [60, 61], and PGRMC1 is thought to be present in the synaptic multiprotein complex bound by A$\beta$o [222, 223].

Understanding the evolutionary origins of TMEM97/PGRMC1 interactions may be instructive in rationalising the system-wide effects of the system, such as roles in disease and pathology. Two regions of PGRMC1 seem to offer the most promising potential TMEM97 interaction sites.

The predicted negatively charged MIHIR alpha helix of Fig. 9C provides a potential interaction site between PGRMC1 and TMEM97. According to the AlphaFold in silico structural prediction [224, 225] and the crystal structure [226] (https://alphafold.ebi.ac.uk/entry/Q5BJF2), the TMEM97 protein contains four transmembrane helices, and a cytoplasmic C-terminal region in solution consisting of residues 162-176 with sequence YKYEEKRKKK. This polybasic sequence could quite conceivably interact electrostatically favourably with the putative MIHIR helix, to the extent that it even has five positive charges to match the five negatives of the MIHIR helix of Fig. 9C, and also C-terminally adjacent negative charges which could interact with the C-terminally adjacent positive charge at position 2 in the MIHIR helix of Fig. 9C. While attractive, this hypothesis requires experimental testing. It predicts that the PGRMC1 MIHIR region is induced into a coiled interaction with another protein that may bind some myosin motor proteins alternatively to PGRMC1, and that the resulting negatively charged exposed helical surface of the PGRMC1 MIHIR interacts with the C-terminus of TMEM97.

Given that the interaction between PGRMC1 and TMEM97 is associated with enhanced endocytosis of the low-density lipoprotein receptor (LDLR) [149, 150], it is tempting to extend the hypothesis to include the proposal that the unknown myosin-binding protein is associated in some way with recruitment of the actin-based motive force required for vesicle internalisation, and that therefore the MIHIR, a eukaryotic MAPR invention [8], is involved with PGRMC1’s membrane trafficking function. Furthermore, Y139 phosphorylation would be predicted to negatively regulate that function.

An alternative candidate region of PGRMC1 to interact with the C-terminal TMEM97 YKYEEKRKKK region is the PGRMC1 C-terminus. The PGRMC1 region from 180-195 (YSDEEEPKDESARKND) has suitable distribution of positively and negatively charged residues for electrostatic interaction. Precisely that region was absent from the PGRMC1 crystal structure [4], indicating a conformationally less restrained region. These alternative possibilities should be studied in the future.

Characterization of the TMEM97 interaction site should be quite informative about PGRMC1 biology. If TMEM97 interacts with the MIHIR, it suggests an ancient biology which predates animals, and could even trace back to the earliest eukaryotes (if the putative TMEM97 interaction does not require phosphorylated PGRMC1 Y139, which evolved in early animals [7]). On the other hand, a TMEM97 interaction with the PGRMC1 C-terminus would indicate biology associated with the evolution leading up to the earliest eumetazoan, since the C-terminus was absent in the common ancestor of earlier branching animal groups such as poriferans and ctenophores, and was only present in the common ancestor of placozoans (which do not have Y180) and eumetazoans (which have Y180) [7].

The LDLR system of lipid transport through the blood is obviously an invention of multi-tissued eumetazoans, such as chordates, with circulatory systems. However, a TMEM97/PGRMC1 mediated mode of vesicular endocytosis could have existed since early eukaryotic times.

In considering the characteristics of the LEUMCA, it is only fitting to turn first to gastrulation, which refers to the embryological invagination of some cells from a topologically ball-like embryo (itself an animal innovation) to generate a cup-shaped intermediate with an outer ectoderm and inner gastroderm (Fig. 11A–C). All animal phyla undergo gastrulation, including sponges, however new cell types do not originate from the formation of the cup like indentation formed by gastrulation [196, 247].

For the sake of completion, note that the diagram depicted for the cnidarian in Fig. 11A–D is certainly an oversimplification. The jellyfish Aurelia appears to undergo a “secondary gastrulation” during metamorphosis from planula larva to polyp, during which larval endoderm is replaced by newly generated tissue [248]. Similar destruction and redevelopment during cnidarian metamorphosis has been observed for nervous system [249, 250], muscles [251] and tentacles [252, 253]. However, consideration of such cnidarian developmental complexity exceeds the purview of this work.

The LEUMCA was the first organism where a differentiated gastroderm persisted to adulthood and was associated with the induction of new patterns of gene expression in some cell types to generate new cell types [247], enabling for the first time a determinated body plan with an extended number of standardized morphological features achieved through a regulated process of cell differentiation. It may be useful to forget the complexity of bilaterian animals and recall that the LEUMCA organizer gave rise to a very primitive organism that would probably be nonviable today, but in its day was the pinnacle of multicellular sophistication. However, it already possessed many of the genes involved in germ layer specification upon which the later bilaterian evolutionary radiation would be based [235].

Importantly, the LEUMCA grade of organization provided an evolutionary springboard, described by Arnellos and Keijzer as “a qualitative jump—a major transition”, from a world of animal movement driven by cilia and contractile epithelia (adherens junction-mediated actin movements) to one of contraction-based (muscular) motility. The organizer was the enabling phenomenon for the evolution of the body forms we find from the very earliest animal fossils of the Ediacaran fauna (Fig. 8C), and later led to the evolutionary extravaganza known as the Cambrian explosion [74], which apparently was also fueled by a period of increased oxygen and biomatter availability compared to Ediacaran times [174].

That this scenario for PGRMC1 function is correct was supported by the recent report of Lee et al. [254] that at ER-plasma membrane junctions PGRMC1 (and also PGRMC2) directly bound to a coiled-coil motif of stromal interaction molecule 1 (STIM1), an EF-hand domain protein which acts as a Ca${}^{2+}$ sensor and is involved in mediating store-operated Ca${}^{2+}$ entry (SOCE) to the cytoplasm from the ER. Prior to SOCE PGRMC1 was in the ER membrane, however, following SOCE a PGRMC1-STIM1 complex was translocated to ER-plasma membrane junction. PGRMC1 depletion reduced SOCE and STIM1 translocation. The formation of SOCE-activated focal adhesion was associated with altered actomyosin cytoskeleton and increased migration [254].

We have previously demonstrated that PGRMC1 phosphorylation mutants affect cell migration [50], and we proposed that the PGRMC1 MIHIR contained a coiled-coiled motif [7] which should be regulatable via Y139 phosphorylation since Y139 constitutes one of the hydrophobic coiled-coil heptad repeat residues (Fig. 10B). Taken together, these results suggest that PGRMC-dependent and regulatable focal adhesion activity via adherens junctions and associated regulated cell migration appeared in the LEUMCA along with and dependent upon a new structure of the PGRMC gene.

Having considered LEUMCA gastrulation, it is imperative that we understand the LEUMCA and its organizer, from which our own branch of the evolutionary tree developed. Descendants of the LEUMCA are called eumetazoans because of their adult possession of a gut and differentiated cell types, including the first neurons (excluding the independent ctenophore development of neuron-like cells) [192]. The group is also sometimes referred to as Gastraea or Gastraeozoa (“animals with an intestine”).

Cnidarian organizer function is not restricted to embryogenesis of a zygote. For instance, in Hydra, pluripotent stem cells are scattered throughout the body. These can permit parthenogenetic formation of new offspring. Even more impressively, a Hydra polyp can also be bisected into multiple fragments (e.g., 20), each of which will grow into a complete adult with all specialized cell types [216]. Therefore, organizer function can arise from multiple stem cells within the Hydra body.

Although the mechanics of gastrulation differ markedly among cnidarian groups [255], it is clear that eumetazoan animals inherited a gastrula life stage from the LEUMCA, and its invention of the organizer [212, 231, 235, 239, 256]. The blastula region on the opposite side to the point of sperm entry becomes the organizer (Cell divisions initiated by formation of the zygote nucleus induce its activity there). The organizer induces epithelial cells to undergo differentiation in a process involving bone morphogenetic protein (BMP) factor and Wnt/$\beta$-catenin signaling, which are required in LEUMCA descendants from cnidarians to mammals to induce expression of the transcription factor Brachyury and organizer function, which leads to downstream activation of the homeobox (hox) transcription factor patterning system [257, 258, 259, 260, 261]. Cnidarians possess individual hox genes, whereas in bilaterians a hox cluster exists [232] (Fig. 11D,G). Nodal signaling is a later, bilaterian, innovation [54]. In animals, Brachyury is essential for gastrulation and the induction of mesoderm. In the cnidarian Hydra, a Wnt/$\beta$-catenin regulated system involving Brachyury drives an organizer activity associated with the hypostome, which is the lip of the gastric cavity, that is responsible for body pattern formation [235, 262].

Brachyury in the LEUMCA, as interpolated from the cnidarian state, activated ectodermal genes, and repressed endodermal genes, and this later evolved into the bilaterian system where triploblasts develop mesoderm from Brachyury-expressing ectoderm [263, 264]. In the early mouse embryo, a structure called the primitive streak forms at mouse embryonic day 6.5, at the time that primed PSCs appear before implantation. Its cells undergo the Wnt/$\beta$-catenin-induced and organizer-driven differentiation process of EMT, where they delaminate from the epithelial layer, become motile, and migrate into the embryo lumen to form mesoderm, in a process dependent upon Brachyury expression (reviewed by [265]). In vertebrates the functions associated with the region called the Spemann-mangold organizer of Xenopus can occur at different times and places in different lineages [266], yet here we are concerned with the origins of the Brachyury-expressing organizer in the LEUMCA, its association with PGRMC, and PGRMC1’s association with Wnt signaling in general cell biology [144] (also discussed above) and particularly in the very early embryo [140].

PGRMC1 and PGRMC2 are expressed in vertebrate oocytes [14, 26, 57, 60, 145, 182, 267, 268, 269, 270, 271, 272, 273, 274] as well as spermatozoa [23, 275], where PGRMC is involved (directly and/or indirectly) with P4 responsiveness (Indeed, this has been one of the most fertile areas of PGRMC1 research, excuse the pun). Importantly, both PGRMC1 and PGRMC2 are expressed from the zygote to blastula stage in mammals [276, 277], and so are present at the onset of gastrulation. In the nematode Ceanorhabbitis elegans the PGRMC homologue Ventral Midline-1 (Vem1) as well as the MAPR NEUFC family member Vem2 are also expressed from the oocyte stage and fertilized zygote, through all developing cell stages to the blastula [234] (Also see Wormbase [278] entries for both proteins: (WBGene00006478#0-9g6e1bcd-10, and WBGene00006890#0-9g6e1bcd-10). We may conclude that the PGRMC gene in the LEUMCA was similarly expressed from zygote to blastula, and therefore was available to participate in organizer determination and activity.

Brachyury is one of the genes that arose during single celled holozoan evolution, which deserves special attention here in the context of metazoan gastrulation and the protozoan phenotypic switching between flagellated and ameboid morphotypes. The filasterean holozoan single-celled organism Capsaspora owczarzaki exhibits dynamically interconvertible histone-modified chromatin states that regulate transcription factor cis-regulatory elements of gene promoters. These are associated with altered gene expression of coregulated genes that characterize different stages of the life cycle. The Brachyury transcription factor is encoded by one of the genes acquired by the common ancestor of Capsaspora and animals. It is a member of the T-box transcription factor family. A T-box protein was present in the ancestor of opisthokonts (the phylogenetic group that contains fungi and animals), but the Brachyury type of T-box protein was first acquired by the ancestor of filastereans, such as Capsaspora [279], and animals.

Capsaspora has a life cycle consisting of three cell morphotypes, one of which is amoeboid and motile [280]. Brachyury induces a program of enhanced actin-dependent amoeboid migration in Capsaspora that involves similar genes used by amoeboid mammalian cell motility [281]. These functions are essential in animal gastrulation and EMT-induced mesoderm formation [282], and the Capsaspora Brachyury protein can at least partially induce gastrulation in Xenopus [279]. Brachyury expression in cancers induces EMT, which is strongly associated with malignancy [283]. It appears that the LEUMCA harnessed suites of genes that had been present for hundreds of millions of years. By the innovation of gastrulation, gene activity was controlled by a novel mechanism of genomic packaging. It is possible that PGRMC1 tyrosine phosphorylation played a foundational role in that packaging mechanism, which deserves to be investigated in the future.

Expression of PGRMC1 in MES-SA uterine sarcoma cells led to EMT [16]. A potential PGRMC1 connection with the Brachyury and its actin cytoskeleton regulation is reinforced by the discovery that the MIHIR of vertebrate PGRMC1 contains a predicted coiled-coil motif related to one found in the coiled-coil region of multiple myosins [7].

As discussed above, Y139 phosphorylation may affect PGRMC1 interactions with the actin cytoskeleton (experimentally unverified). Furthermore, in human cell culture the mutation of presumed negative regulatory S57 and S181 residues (both adjacent to Y180 in the folded protein structure [66]) caused increased cell migration in scratch motility assays, and elevated expression of a set of proteins associated with actin cytoskeleton. This was associated with activation of the PI3K/Akt pathway, and efficient subcutaneous mouse xenograft tumor formation. Upon additional mutation of Y180 the elevated actin cytoskeletal proteins, the enhanced motility, the PI3K activation, and efficient xenograft tumor formation were all absent [50]. The effects appear to have been mediated by altered PGRMC1-dependent genomic epigenetic gene packaging [19].

Since 2012 we have known that PGRMC1 could affect gene transcription, since its depletion resulted in elevated expression of a large number of genes [143]. It is likely that epigenetics was involved, however Sumoylated PGRMC1 directly binds and activates promoters with binding sites for the transcription factor T-cell factor/lymphoid enhancer-binding factor (TCF/LEF), a target of the Wnt pathway [142, 143, 144]. Taken together, these results suggest that PGRMC Y180 phosphorylation could regulate or is involved in Brachyury function during organizer function, which merits further investigation.

We have also recently demonstrated that PGRMC1 is found in immune precipitation complexes with many proteins associated with the actin cytoskeleton, and that the small molecule inhibitor AG-205 prevents many of those interactions [91]. Salsano et al. [172] also identified many proteins associated with actin cytoskeleton and membrane trafficking to be co-precipitated with PGRMC1. However, it remains to be demonstrated that actin interactions are via the MIHIR region. Saliently, PGRMC1 affects both actin cytoskeletal protein abundances and cell motility or migration [50], many instances of which are associated with EMT and/or tumor metastasis [16, 50, 284, 285, 286, 287, 288, 289].

A working hypothesis emerges where PGRMC-dependent alterations in actin cytoskeletal regulation during EMT modulate adherens junction-mediated epithelial cell adhesion with accompanying enhanced cell motility, enabling the epithelial to mesenchymal transition that underpins bilaterian evolution, and the development of mesoderm. In other words, the acquisition of PGRMC1 Y139 and Y180 by the LEUMCA may have facilitated gastrulation, and the evolution of eumetazoans.

However conjectural this may be, the hypothesis predicts that PGRMC1 may regulate proteins of the adherens junction. PGRMC1 phosphorylation status can modulate vinculin abundance [50], and the small molecule AG-205 disrupts protein complexes involving PGRMC1 and proteins of the actin cytoskeleton (which contained calponin homology (CH) actin-binding domains), among which $\alpha$-actinin-1 was the most significantly affected [91]. Both vinculin and $\alpha$-actinin are components of adherens junctions [290, 291, 292] (Fig. 10).

The $\alpha$-actinins (member of the spectrin family) are actin filament bundling proteins which diversified during vertebrate evolution. They function to both cross link actin microfilaments and anchor them to other subcellular structures, which was probably critical to the origin of effective actomyosin-based generation of force. The $\alpha$-actinin family diversified with the development of different muscle types. Invertebrates typically have one gene of $\alpha$-actinin, whereas vertebrates typically have three or more genes. A general $\alpha$-actinin functional theme involves cooperation with myosin II motors in contractile systems associated with diverse functions including cytokinesis, muscle contraction, and cell motility [293]. Muscular contraction, and the acto-myosin-driven motility of an elongating neural axon, both arose along with the gastrulation organizer in the LEUMCA. The bilaterian (co-temporal with acquisition of PGRMC T178 and S181; Fig. 8B) gastrulation process additionally involves the interruption of adherens junction-mediated cell contact and the actomyosin-mediated migration of epithelial cells into the embryo interior (Fig. 11E). Taken together, it is reasonably likely that the PGRMC function modified by the acquisition of Y139/Y180 in the LEUMCA affected adherens junctions and the regulated aggregation/disaggregation of actin microfilaments into stress fibers, which may have facilitated the following biology. Once more, this is conjecture.

The LEUMCA was the first animal where the embryological gastrula stage persisted to adulthood as a specialized gut, having spawned a suite of different specialized cell types in the process [247]. The gastro-epithelial surface is thought to have developed muco-ciliary secretions and to have been folded into gastric pouches which optimized nutrient absorption and permitted extracellular digestion [294]. This created a novel environment for bacterial colonization. Bacteria subsequently played a crucial part in the evolution of animals, with specialized microbial communities colonizing ectoderm and gut endoderm of cnidarians (reviewed by [295]) and triploblastic bilaterians. In the latter the gut forms a microbe-filled tube where bacteria ferment digested food into products that can be used by the host animal under anoxic conditions from worms [296] to mammals [297]. That coevolutionary journey between bacteria of the microbiome and host animals was born with the LEUMCA and its development of the specialized gut.

In mammals, colonic epithelial cells (colonocytes) consume oxygen by oxidative phosphorylation to promote gut lumen hypoxia associated with obligate anaerobic healthy gut microbiota. Failure to maintain hypoxic levels permits the expansion of facultative anaerobes, leading to dysbiosis that is associated with several pathologies [297]. In this process, the progenitor crypt stem cells perform Warburg glycolytic metabolism. Recall that PGRMC1 directs Warburg glycolytic metabolism [49, 50]. The differentiation process of mammalian gut epithelium involves a metabolic switch to activation of fatty acid catabolism and oxidative phosphorylation by mature colonocytes. Therefore, glucose metabolism is crucial in healthy gut function [297], and this dichotomous gut cell metabolism may be inherited from the LEUMCA. This sounds like a system that (hypothetically) could have been driven by PGRMC since LEUMCA times. We will consider glandular cells of the gut in a separate section.

Innervated striated muscles are present in both cnidarians and bilaterians, however these have different architectural organization and have been shown to have evolved independently in both lineages from a pre-existing suite of epitheliomuscular LEUMCA contractile apparatus [298]. We must conclude that the LEUMCA is not likely to have possessed striated muscle. Cnidarian muscle is innervated by neurons belonging to a nerve net with sensory neurons that enable rapid detection of and response to environmental stimuli that is superior to those of earlier-branching animals [74] (We will deal with neurons below).

However, the biology of the LEUMCA and its descendants is intimately associated with contraction-based motility that enabled predation of food that utilized the new gastric cavity [74]. The LEUMCA was probably benthic (sea floor-dwelling) and used cilia and mucous to trap organic material [247, 294]. It is then perhaps best to imagine coordinated organism-wide cellular epitheliomuscular contractions not yet at the level of striated muscle but providing sufficient locomotion to successfully forage for food. Cnidarians generate peristaltic waves via radial muscle contractions based upon coordinated adherens junction contractions [299]. The earliest known identifiably bilaterian fossil is thought to have burrowed through shallow nutrient-rich ocean sediments using peristaltic motion [230], which would have required a system of longitudinal and transverse muscle-like fibers.

Insulin-related peptides are conserved in deuterostomian, ecdysozoan, and lophotrochozoan bilaterian species, as well as cnidarians [300]. Therefore, they can be extrapolated back to the LEUMCA. The reconstructed LEUMCA possessed insulinergic glandular cells expressing the forkhead box transcription factor A (FoxA), which gave rise to related cnidarian (Fig. 12B, Ref. [216]) and mammalian (pancreatic) gland cells [227, 228, 229]. Insulin controls glucose homeostasis. PGRMC1 is known to regulate plasma membrane translocation of the insulin receptor, glucose transporters GLUT1 and GLUT4 [301], as well as glucagon-like peptide-1 (GLP1) receptor (GLP1R) [302], all of which are associated with hormonal glucose homeostasis. 3T3L1 pre-adipocyte stem cells also upregulate glucose intake by PGRMC1-dependent translocation of GLUT4 to the plasma membrane, where PGRMC1 and GLUT4 are co-localized and can be co-immunoprecipitated [162].

Mammalian GLP1 is a peptide derived from the pro-glucagon precursor. This precursor protein encodes several different and in part overlapping incretin proteins which are generated by differential proteolytic cleavage, including glicentin, glicentin-related polypetide, oxyntomodulin, glucagon, GLP1 and GLP2 [303] (And see UniProt P01275). While glucagon is produced by pancreatic A cells under low blood glucose conditions, GLP1 is produced post-prandially by intestinal enteroendocrine L cells, pancreatic A cells, and the central nervous system. A hypothesis can be proposed here that this system represents an evolutionary descendant of PGRMC functions from the LEUMCA that mediated communication between cell types similar in complexity and spatial organization to those of Fig. 12B.

Oral glucose administration evokes a much stronger insulin release, due to intestinal GLP1 release, than is evoked by intravenous glucose injection. GLP1 acts on the pancreas to induce insulin secretion and on the central nervous system leading to appetite reduction (for reviews: [303, 304, 305]). As such, PGRMC is positioned to regulate energy communication between cells of bilaterian animals, which is possibly inherited from LEUMCA glandular cells.

This is a hypothetical proposal, so far undemonstrated. Future research should address the evolutionary history of the association of PGRMC and its Y139/Y180 phosphorylation residues with this system. However, it is notable that PGRMC1 also regulates glucose metabolism on a cellular level in response to P4, seemingly independently of glucagon-insulin signaling (although this has not been formally examined) [49, 306] and glycolytic metabolism is affected by PGRMC1 phosphorylation [50].

One relationship between glucose metabolism and the new LEUMCA requirement for synaptic function could be due to membrane fluidity. Here, we need to briefly introduce the fact that a class of serpentine seven membrane-spanning G protein-coupled receptors are associated with membrane P4 receptor activity. A complex involving PGRMC1, PGRMC2 and PAQR7 (or mPR$\alpha$, see above) was responsible for mediating P4 responses in human granulosa/luteal cells [59]. In zebrafish, PGRMC1 regulation of the cell surface localization of PAQR7 led to a PAQR7-mediated P4 response [307].

PGRMC1 also regulates the cell surface availability of a variety of other known proteins [47], including the insulin receptor [301]. What has this to do with membrane fluidity? PAQR-2 and insulin growth factor receptor-like 2 (IGLR-2) modulate membrane fluidity in response to glucose levels by regulating fatty acid desaturation levels in nematodes [308] and mammals [309]. Membrane fluidity is important for synaptic vesicle trafficking, as well as for the cell surface localization of receptors involved in ligand-dependent guidance of cell movement. PGRMC1 is implicated in fatty acid synthesis [41, 162, 163], and both PGRMC1 and PGRMC2 affect the activity of fatty acid 2-hydroxylase that is required for the synthesis of 2-hydroxylated sphingolipids in the nervous system and other cell types, which has been proposed to reflect PGRMC-dependent functional interdependence of sphingolipid and sterol biology on membrane properties [164]. It will be interesting to see whether PGRMC1 is involved in this possible modulation of membrane fluidity in response to insulin/glucagon hormonal regulation that could have been inherited from the LEUMCA.

Insulin affects synaptic function and the plasticity required for long-term potentiation [310], and pronounced hippocampal glucose consumption has long been known to be associated with learning and memory formation [311]. Accordingly, impaired glucose metabolism is associated with several neurodegenerative conditions [312, 313]. Therefore, regulation of glucose metabolism by PGRMC could have been associated with the evolution of the nervous system and ligand-guided cell migration. Having now been diverted from endocrine regulation of metabolism towards neural function, it is appropriate to consider neurons in more detail.

Sleep is critical for neural function and memory formation [361, 362]. During sleep some synapses are weakened and others strengthened to consolidate recent memories in the form of neural excitation pathways [363].

Of relevance to our theme, synaptic changes occur during sleep, and sleep evolved at the same time as the organizer, i.e., in the LEUMCA. Cnidarians change behavior at night, which is considered as sleep because animals exhibit slower reaction times in this state, and if deprived they will later ‘sleep’ longer to catch up: a behavior known as homeostatic sleep regulation. This implies that sleep is related to a circadian-associated neural regeneration process that appeared before highly centralized nervous systems [364, 365, 366]. For the moment, note that sleep appeared evolutionarily at the same time as the gastrulation organizer, Netrin and DCC, the first neurons, and the combination of PGRMC Y139 and Y180.

Also recall that PGRMC2 knockout led to reduced heme shuttling to the nucleus, which led to increased stability of heme-responsive transcriptional repressor Rev-Erb$\alpha$, also known as nuclear receptor subfamily 1 group D member 1 (NR1D1), the homeostatic negative feedback regulator of heme biosynthesis, and impaired ability of brown fat mitochondria to produce thermogenesis [82]. Strikingly, in the context of this section, Rev-Erb$\alpha$ is a heme receptor that not only coordinates metabolism via mitochondrial function (a hypothesized eukaryogenic ancestral PGRMC function), but also circadian rhythm related to sleep [367]. That observation represents either coincidence or provides evidence in favor of the hypothesis.

As a working hypothesis, we could do worse than propose that the coincident appearance of gastrulation, PGRMC tyrosine phosphorylation at Y139 and Y180, muscles, synapsed nerves, and photoreceptors, together with Hif-1, the Netrin/DCC (and Netrin/Neogenin) system and ‘sleep’ (diurnal behavioral changes) were all part of the emergent biology of the LEUMCA, dependent in good part on PGRMC tyrosines. If the LEUMCA migrated to the bright upper water layers by day to ingest the plentiful photosynthetic organisms there, they would have been localized by photodetection, hunted by neuronally-directed muscle motor propulsion, and transferred to a new gut. There, specialized epithelial cells enhanced digestion and absorption, probably assisted by the very first specialized gut microbiome, as the organism ‘slept’ to regenerate. It may also have been benthic, sifting through seabed detritus. This organism may have been primitive by modern standards, but its attributes would transform life on earth.

Conventional thought has attributed sleep regulation in insects to monoamine and peptide neurotransmitters [368], however it has long been known that the insect steroid ecdysone regulates sleep in the fruit fly Drosophila melanogaster [369]. It will be interesting to see whether Drosophila PGRMC (called membrane steroid binding protein, MSBP: UniProt Q9VXM4) regulates ecdysone production and sleep. The production of several reproductive hormones is affected by sleep and circadian rhythm in humans, which influences fertility (another area of PGRMC ‘core biology’). Likewise, reduced steroid levels lead to poor quality of sleep [370]. We will return to sleep and memory formation in consideration of gut flora below, and Alzheimer’s disease and circadian NAD (nicotinamide adenine dinucleotide) metabolism in the accompanying paper [2].

In the transition from earlier-branching animals such as ctenophores and poriferans to the LEUMCA, the PGRMC gene gained a C-terminal extension which contains the Y180 motif (Fig. 8B). Tsai and colleagues [371] from the University of Michigan Medical School have recently demonstrated that this region participates in a newly described function for PGRMC1: binding to low molecular weight complexes of misfolded proteins in the lumen of the ER and targeting them for ER-phagy (literally ‘ER eating’) via the reticulon-3 (RTN3)-driven targeting complex in HEK293 cells, Cos cells, and ex vivo primary pancreatic islets. In ER-phagy, portions of the ER are portioned off and sent to the endolysosome for degradation (for review: [372]). Notably, in this function PGRMC1 has the type 2 membrane topology observed in embryonic stem cells [373], Alzheimer’s neuron synapses [64], hepatocytes [374], and cultured lung cancer cells [375]. That is, the C-terminus is in the ER lumen, and hence unavailable to cytoplasmic enzymes such as kinases, ubiquitin conjugating enzymes, and the majority of CYP450 molecules that have cytoplasmic active sites [376]. The PhosphositePlus site contains hundreds of references of PGRMC1 with post-translational modifications of the C-terminus consistent with a type 1 orientation (https://www.phosphosite.org/proteinAction.action?id=5744). It remains unclear why PGRMC1 is found with different membrane topologies in different circumstances. The answer will probably illuminate a deep truth about eukaryotic biology.

While ctenophores, poriferans and placozoans have paraHox genes, cnidarians and bilaterians possess Hox genes which specify anterior-posterior identity during embryogenesis. There is no evidence for a Hox cluster in cnidaria. Hox clusters evolved in bilaterian animals, and exhibit the principle of spatial collinearity, where their expression along the anterior-posterior axis of the embryo corresponds to the order of genes within the cluster [232]. There is no apparent link between PGRMC and this LEUMCA innovation, but the hox cluster was instrumental in the subsequent evolution of bilaterians.

At the outset of this section, it must be strongly stated that the author is unaware of any evidence that associates PGRMC1 with germline segregation. This section is being included because PGRMC1 is related to steroid responses of the mammalian male and female reproductive systems (i.e., germline cells), and because germline segregation appeared around the time that PGRMC1 acquired its phosphorylated tyrosines Y139 and Y180 in the LEUMCA, with especially the adjacent T178 and S181 regulatory residues in bilaterians that undergo germline segregation.

Very briefly, impregnation of the oocyte by the sperm forms the zygote. As soon as maternal genes are switched off and embryonic transcription commences, epigenomic reprogramming occurs, removing methyl groups from histones and DNA, to generate a hypomethylated ‘clean slate’ epigenetic state. The resulting cells are said to be in a “naïve” state of pluripotency and are called naïve PSCs. These can transition through a variety of epigenetic and metabolic intermediate pluripotential states, until they undergo genomic hypermethylation to form “primed” PSCs, which can then differentiate into multiple lineages. Primordial germ cells differentiate from one of the intermediate pluripotency states. Great advances in our understanding of this process at a molecular level have been made over recent years, to the extent that the process can be directionally steered in vitro to generate fully functional oocytes or spermatogonia [403, 404, 405].

Newman [195] reviews how differentiated animal cell specializations probably co-opted suites of genes that had been co-regulated in their unicellular ancestors. In single celled organisms, histone modifications had been utilized to elevate or suppress gene expression since the first eukaryotes. Metazoans including poriferans invented upstream or downstream enhancer regions whose transcription factor binding sites resembled those of gene promoters proximal to transcription start sites, but which could regulate the expression levels of large regions of chromosome [195].

Enhancers permitted cells to switch on or off parts of the chromosome so that cells with a single genome could exhibit totally different phenotypes [195]. Bilaterian embryology is associated with substantial changes in the three-dimensional organization of the genome because of epigenetic changes [411, 440].

Transcriptionally active gene activity can be up- or down-regulated by modulated protein binding to associated transcriptional enhancers, which are cis-acting genomic sequences with specific transcription factor binding sites [441]. Enhancer activity, especially in vertebrates, is regulated by methylation of cytosine residues adjacent to guanosine in the DNA sequence at so-called CpG (cytosine-phosphodiester-guanosine) motifs. In vertebrates CpG motifs have been negatively selected and are largely located in “islands” found in promoters and to a lesser extent enhancers [442]. DNA methylation at CpG motifs is ancient in eukaryotes, although it can be absent from specific species, such as the yeast S. cerevisiae, and the nematode C. elegans [443].

While CpG methylation of promoter and enhancer elements is most often associated with transcriptional silencing, demethylation itself is not sufficient for transcriptional activity, and the rules related to individual enhancer activity remain poorly understood [409]. Once this mechanism existed in the ancestor of the LEUMCA, the fuel for eumetazoan evolutionary diversity was available, apparently awaiting only the acquisition of a gastrulation organizer, lineage determining transcription factors, novel tissue-specific proteins, and sufficient time for natural selection to act to produce the hierarchical cascade such as vertebrate embryogenesis (or perhaps more incredibly, the larval, pupal metamorphosis, and adult life cycle stages of insects).

The rest, as they say, is evolutionary history. However, this is relevant to PGRMC phosphorylation because this process is/was initiated by the gastrulation organizer (which coincided evolutionarily with the acquisition of PGRMC1 Y139 and Y181), and mutation of PGRMC1 phosphorylation sites causes dramatic changes in genomic CpG methylation state of cancer cells [19]. Gastrulation itself requires enhancer demethylation in mice [444], and thousands of enhancers are coordinately demethylated during early vertebrate embryogenesis in the phylotypic stage, the early embryogenic stage when different taxa are morphologically similar [445].

Primed induced PSC (iPSC) formation from somatic cells involves a net increase in the level of genomic CpG, accompanied by the active demethylation of specific pluripotency-associated enhancers [409]. Mouse embryonic stem cells cultured in media along with feeder cells, or supplemented with leukemia inhibitory factor (LIF), exhibit similar hypermethylation of the genome. However, culturing embryonic stem cells in serum free media that contains two kinase inhibitors—targeting mitogen-activated protein kinase kinase (MEK) and GSK-3$\beta$—produces a hypomethylated genome that is thought to recapitulate the naïve pluripotent ground state [446, 447]. In the developing mammalian embryo, the zygote genome is hypomethylated, but becomes hypermethylated by the time of implantation (E6.5 and E7.5 in the mouse) [200, 448]. A state of hypermethylation of most genes, and hypomethylation of selected promoters, occurred when we mutated Y180 in cancer cells. Pathways analysis of epigenetically affected genes suggested processes related to early embryonic development were being affected [19]. There is therefore a strong association between the biology of PGRMC1 Y180 observed in cultured cancer cells, and the embryological epigenetic modifications of the genome initiated following the activity of the gastrulation organizer. While it is not clear that tyrosine-phosphorylated PGRMC1 contributed to the origin of the organizer, the case therefore is captivating.

Note here that pluripotent embryonic stem cells represent the pre-gastrulation stage of development, since they can be induced to form gastrulating embryoid bodies [449, 450]. When we mutated PGRMC1 Y180 [19], whose evolutionary appearance coincided with the appearance of gastrulation [7], the cells superficially converted to a hypermethylated state resembling the genomic hypermethylation level of primed embryonic stem cells, representing the pre-gastrulation blastula. This is reminiscent of the report from David Sinclair and colleagues that ectopic expression of transcription factors Oct4, Sox2 and Klf4 in retinal ganglion cells led to a restoration of youthful DNA methylation patterns, which was accompanied by a revitalization of neural functions [451]. These are the three of the same transcription factors previously shown by Takahashi and Yamanaka to revert somatic fibroblasts to iPSCs [452]. The novelty of the Sinclair study’s findings was that gene dosage can induce partial reversion from sub-optimal/diseased state back to correctly differentiated state, without promoting total dedifferentiation and stem cell characteristics with associated risks such as increased cancer propensity [451]. The author predicts that Oct4, Sox2 and Klf4 will cause PGRMC1 post-translational status as part of their reprogramming mechanism of action.

In line with the hypothesis that the pleiotropic effects of PGRMC1 on cell differentiation state are related to epigenetics and genomic status, Pedroza et al. [453] profiled the miRNome of MDA-MB-468 triple negative breast cancer cells after AG-205 treatment to inhibit PGRMC1 function (although they do not acknowledge that AG-205 has off-target effects [90], see Section 2.1.1 above) or anti-PGRMC1 siRNA transfection to reduce PGRMC1 levels.

This was the first study to compare attenuation of PGRMC1 with the effects of AG-205 treatment. Although the miRNAs most strongly altered by AG-205 and PGRMC1 siRNA were non-overlapping (1008 miRNAs following AG-205 treatment and 776 miRNAs after PGRMC1 siRNA), Pedroza et al. [453] discussed the results broadly under PGRMC1 effects, with the clear assumption that all AG-205 effects are mediated through PGRMC1. For instance, in the results description of AG-205 treatment, the section heading begins with “PGRMC1 Signal Disruption Alters miRNAs…”. The discussion section makes no discrimination between AG-205 and siRNA PGRMC1, conflating both sets of results into one dialogue. From the results descriptions, Kyoto Encyclopedia of Genes and Genomes (KEGG) terms of most affected miRNA-affected pathways for AG-205 and PGRMC1 siRNA treatment involved p53 signaling pathway, cell cycle and pathways in cancers, albeit via different miRNAs and affected genes [453]. There is no data presented which shows that AG-205 exerts only PGRMC1-specific effects, and non-specific effects were not considered by the authors. The degree to which AG-205 effects are due to PGRMC1 requires urgent evaluation.

More interestingly in the context of the current discussion, by considering only the PGRMC1 siRNA results, we can conclude that the presence of PGRMC1 is required for the persistent expression status of hundreds of regulatory miRNAs (to either maintain or repress transcription, respectively). One way in which that could be accomplished would be by PGRMC1 participation in a mechanism responsible for epigenetic genomic maintenance, as we observed in MIA PaCa-2 pancreatic cancer cells expressing PGRMC1 phosphorylation site mutants [19]. Pathways associated with cell cycle and cancers can be considered as surrogates for dedifferentiation. This is a testable hypothesis, which predicts that the AG-205 and PGRMC1 siRNA treatments will exhibit induced epigenetic changes in the MDA-MB-468 cell system of Pedroza et al. [454].

In this respect, the report of Kim et al. [140] that PGRMC1 maintains primed PSC pluripotency seems extremely relevant. They reported that PGRMC1 knockdown inhibited GSK-3$\beta$ leading to Wnt signaling via $\beta$-catenin, which is precisely the pathway activated by gastrulation. It is unlikely to be coincidence that sumoylated PGRMC1 is nuclear and can directly bind to TCF/Lef transcription factor sites [142, 143, 144], which are the target of Wnt/$\beta$-catenin signaling as discussed above. Intriguingly, Kim et al. [140] found that the C-terminus of PGRMC1 in type 2 membrane orientation was present on the surface of stem cells, which is opposite to the conventional type 1 cytoplasmic orientation (Fig. 1A) (which would be required for modification by cytoplasmic enzymes such as kinases), but corresponds to the orientation observed on the synaptic surfaces of Alzheimer’s neurons [64].

A previous study by Sperber et al. [455] had reported that stem cell pluripotency was maintained by the production of the metabolite 1-methylnicotinamide (1-MNA) by the enzyme nicotinamide-N-methyltransferase transferase (NMNT). They proposed that consumption of S-adenosyl methionine (SAM) in this reaction pleiotropically reduced the levels of available SAM methyl donor, and so pleiotropically the level of histone methylation, leading to the induction of the Wnt pathway [455]. Note that Kim et al. [140] concluded that stem cell pluripotency of primed PSCs was affected by PGRMC1, while Sperber et al. [455] concluded that NNMT activity maintained the naïve state. We found that PGRMC1 Y180 phosphorylation status modulated NNMT levels, but we observed no effects on SAM levels while we observed altered genomic CpG methylation [19]. While our study was not performed in stem cells, the pathways involved are consistent with PGRMC1 potentially being responsible for modulating the genomic epigenetic landscape of pre- and post-gastrulation cells. This theme is discussed in terms of aging biology in the accompanying paper [2].

Finally, the seemingly unrelated biology of sumoylated PGRMC1 binding the TCF/Lef sites [142, 143, 144], GSK-3$\beta$ regulation of glycogen synthesis and epithelial-mesenchymal transition at the root base of eumetazoan evolution, and their regulation by PGRMC, as well as PGRMC control of actin cytoskeleton, migration, axon guidance, vesicle-mediated cell-cell communication, and cell metabolism, may all be reconciled if we consider the relatively primitive body grade and requirements of the original LEUMCA. These may have all been functions that the offspring of a blastula-stage epithelial cell would have to differentially regulate within a few cell divisions following the activity of the gastrulation organizer. The fact that we observe these functions in different parts of the mammalian body may obscure the fact they were ancestrally part of responses that were spatially and temporally proximal to each other in a very simple organism (Fig. 11), which probably had to make relatively unsophisticated environmentally-driven phenotypic changes to cell function, in contrast to the complexity of mammalian embryology.

As the mammalian embryo progresses from zygote to blastula, and begins to prepare for gastrulation, the parental CpG methylation pattern is reset to generate a hypomethylated state that corresponds to naïve PSCs. This progresses to the hypermethylated state of primed PSCs around the time of gastrulation (E6.5/E7.5 in the mouse). While most CpG sites in the genome are methylated, a relatively smaller number of sites are hypomethylated. A number of these hypomethylated sites become progressively methylated during the lifespan, which can be used to measure biological age [456]. This mechanism, which has become known as the Horvath clock, is conserved across mammalian species [457, 458, 459].

Of extreme relevance to this section, the aging clock is reset to zero around the time of gastrulation. Cultured PSCs do not age, and the methylation clock begins once primed PSCs begin to differentiate, or when differentiation processes are induced by gastrulation [460]. PGRMC1 obtained its tyrosines in evolution as the same time as the gastrulation organizer [7], and mutation of Y180 in a cancer cell line induced a hypermethylated state reminiscent of primed PSCs [19]. The primed type of PSCs most probably evolved with the LEUMCA (PGRMC1 Y139 and Y180), or the urbilaterian (PGRMC1 T178 and S181), at a time when more highly differentiated cell types did not yet exist, and the metabolic switch was related to some aspect of the survival of an animal that was relatively simple by modern standards.