Peptide name : Sigma intracellular receptor 2

Source/Organism : Human

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 176

C-terminal modification: Not found

N-terminal modification : Not found

Non-natural peptide information: None

Assay type : Antibody-based assay

Assay time : 48h

Activity : Not found

Cell line : HaCat

Cancer type : Not specified

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 20847.6229 Dalton

Aliphatic index : 1.014

Instability index : 50.2176

Hydrophobicity (GRAVY) : 0.2165

Isoelectric point : 9.4217

Charge (pH 7) : 7.7434

Aromaticity : 0.176

Molar extinction coefficient (cysteine, cystine): (36900, 37025)

Hydrophobic/hydrophilic ratio : 1.41095890

hydrophobic moment : 0.0805

Missing amino acid : None

Most occurring amino acid : L

Most occurring amino acid frequency : 27

Least occurring amino acid : N

Least occurring amino acid frequency : 1

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.1, 0.4)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCSC)[C@@H](C)O)C(C)C)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)C(C)C)C(C)C)C(C)C)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)CC)C(C)C)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)CC

1 : Gerhard DS, et al. The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome Res. 2004; 14:2121-7. doi: 10.1101/gr.2596504

2 : Son KN, et al. Histatin-1 is an endogenous ligand of the sigma-2 receptor. FEBS J. 2021; 288:6815-6827. doi: 10.1111/febs.16108

3 : Burkard TR, et al. Initial characterization of the human central proteome. BMC Syst Biol. 2011; 5:17. doi: 10.1186/1752-0509-5-17

4 : Zody MC, et al. DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage. Nature. 2006; 440:1045-9. doi: 10.1038/nature04689

5 : Sanchez-Pulido L and Ponting CP. TM6SF2 and MAC30, new enzyme homologs in sterol metabolism and common metabolic disease. Front Genet. 2014; 5:439. doi: 10.3389/fgene.2014.00439

6 : Alon A, et al. Identification of the gene that codes for the σ₂ receptor. Proc Natl Acad Sci U S A. 2017; 114:7160-7165. doi: 10.1073/pnas.1705154114

7 : Pati ML, et al. Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1. Pharmacol Res. 2017; 117:67-74. doi: 10.1016/j.phrs.2016.12.023

8 : Ma D, et al. GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization, mitochondria-targeting and -activating properties of human salivary histatin 1. Int J Oral Sci. 2022; 14:42. doi: 10.1038/s41368-022-00181-5

9 : Kayed H, et al. Expression analysis of MAC30 in human pancreatic cancer and tumors of the gastrointestinal tract. Histol Histopathol. 2004; 19:1021-31. doi: 10.14670/HH-19.1021

10 : Bartz F, et al. Identification of cholesterol-regulating genes by targeted RNAi screening. Cell Metab. 2009; 10:63-75. doi: 10.1016/j.cmet.2009.05.009

11 : Cheng YS, et al. A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes. Nat Chem Biol. 2021; 17:1271-1280. doi: 10.1038/s41589-021-00907-2

12 : Thejer BM, et al. Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97. Int J Mol Sci. 2023; 24:(unknown pages). doi: 10.3390/ijms24076381

13 : Murphy M, et al. Identification and characterization of genes differentially expressed in meningiomas. Cell Growth Differ. 1993; 4:715-22.

14 : Ahmed IS, et al. S2R(Pgrmc1): the cytochrome-related sigma-2 receptor that regulates lipid and drug metabolism and hormone signaling. Expert Opin Drug Metab Toxicol. 2012; 8:361-70. doi: 10.1517/17425255.2012.658367

15 : Alon A, et al. Structures of the σ₂ receptor enable docking for bioactive ligand discovery. Nature. 2021; 600:759-764. doi: 10.1038/s41586-021-04175-x

16 : Ota T, et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet. 2004; 36:40-5. doi: 10.1038/ng1285

17 : Guo L and Zhen X. Sigma-2 receptor ligands: neurobiological effects. Curr Med Chem. 2015; 22:989-1003. doi: 10.2174/0929867322666150114163607

18 : Wilcox CB, et al. Coordinate up-regulation of TMEM97 and cholesterol biosynthesis genes in normal ovarian surface epithelial cells treated with progesterone: implications for pathogenesis of ovarian cancer. BMC Cancer. 2007; 7:223. doi: 10.1186/1471-2407-7-223

19 : Vaca Jacome AS, et al. N-terminome analysis of the human mitochondrial proteome. Proteomics. 2015; 15:2519-24. doi: 10.1002/pmic.201400617

20 : Huang YS, et al. Sigma-2 receptor ligands and their perspectives in cancer diagnosis and therapy. Med Res Rev. 2014; 34:532-66. doi: 10.1002/med.21297

21 : Ebrahimi-Fakhari D, et al. Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells. Hum Mol Genet. 2016; 25:3588-3599. doi: 10.1093/hmg/ddw204

22 : Riad A, et al. Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex. Sci Rep. 2018; 8:16845. doi: 10.1038/s41598-018-35430-3

Paper title : The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

Doi : https://doi.org/10.1101/gr.2596504

Abstract : The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Paper title : Histatin-1 is an endogenous ligand of the sigma-2 receptor.

Doi : https://doi.org/10.1111/febs.16108

Abstract : The Sigma-2 receptor (S2R) (a.k.a TMEM97) is an important endoplasmic reticular protein involved in cancer, cholesterol processing, cell migration, and neurodegenerative diseases, including Niemann-Pick Type C. While several S2R pharmacologic agents have been discovered, its recent (2017) cloning has limited biological investigation, and no endogenous ligands of the S2R are known. Histatins are a family of endogenous antimicrobial peptides that have numerous important effects in multiple biological systems, including antifungal, antibacterial, cancer pathogenesis, immunomodulation, and wound healing. Histatin-1 (Hst1) has important roles in epithelial wound healing and cell migration, and is the primary wound healing agent in saliva. Little is understood about the downstream machinery that underpins the effects of histatins, and no mammalian receptor is known to date. In this study, we show, using biophysical methods and functional assays, that Hst1 is an endogenous ligand for S2R and that S2R is a mammalian receptor for Hst1.

Paper title : Initial characterization of the human central proteome.

Doi : https://doi.org/10.1186/1752-0509-5-17

Abstract : BACKGROUND: On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses. RESULTS: We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation. CONCLUSIONS: Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.

Paper title : DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage.

Doi : https://doi.org/10.1038/nature04689

Abstract : Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

Paper title : TM6SF2 and MAC30, new enzyme homologs in sterol metabolism and common metabolic disease.

Doi : https://doi.org/10.3389/fgene.2014.00439

Abstract : Carriers of the Glu167Lys coding variant in the TM6SF2 gene have recently been identified as being more susceptible to non-alcoholic fatty liver disease (NAFLD), yet exhibit lower levels of circulating lipids and hence are protected against cardiovascular disease. Despite the physiological importance of these observations, the molecular function of TM6SF2 remains unknown, and no sequence similarity with functionally characterized proteins has been identified. In order to trace its evolutionary history and to identify functional domains, we embarked on a computational protein sequence analysis of TM6SF2. We identified a new domain, the EXPERA domain, which is conserved among TM6SF, MAC30/TMEM97 and EBP (D8, D7 sterol isomerase) protein families. EBP mutations are the cause of chondrodysplasia punctata 2 X-linked dominant (CDPX2), also known as Conradi-Hünermann-Happle syndrome, a defective cholesterol biosynthesis disorder. Our analysis of evolutionary conservation among EXPERA domain-containing families and the previously suggested catalytic mechanism for the EBP enzyme, indicate that TM6SF and MAC30/TMEM97 families are both highly likely to possess, as for the EBP family, catalytic activity as sterol isomerases. This unexpected prediction of enzymatic functions for TM6SF and MAC30/TMEM97 is important because it now permits detailed experiments to investigate the function of these key proteins in various human pathologies, from cardiovascular disease to cancer.

Paper title : Identification of the gene that codes for the σ₂ receptor.

Doi : https://doi.org/10.1073/pnas.1705154114

Abstract : The σ₂ receptor is an enigmatic protein that has attracted significant attention because of its involvement in diseases as diverse as cancer and neurological disorders. Unlike virtually all other receptors of medical interest, it has eluded molecular cloning since its discovery, and the gene that codes for the receptor remains unknown, precluding the use of modern biological methods to study its function. Using a chemical biology approach, we purified the σ₂ receptor from tissue, revealing its identity as TMEM97, an endoplasmic reticulum-resident transmembrane protein that regulates the sterol transporter NPC1. We show that TMEM97 possesses the full suite of molecular properties that define the σ₂ receptor, and we identify Asp29 and Asp56 as essential for ligand recognition. Cloning the σ₂ receptor resolves a longstanding mystery and will enable therapeutic targeting of this potential drug target.

Paper title : Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1.

Doi : https://doi.org/10.1016/j.phrs.2016.12.023

Abstract : A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued.

Paper title : GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization, mitochondria-targeting and -activating properties of human salivary histatin 1.

Doi : https://doi.org/10.1038/s41368-022-00181-5

Abstract : Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Paper title : Expression analysis of MAC30 in human pancreatic cancer and tumors of the gastrointestinal tract.

Doi : https://doi.org/10.14670/HH-19.1021

Abstract : Meningioma-associated protein, MAC30, is a protein with unknown function and cellular localization that is differentially expressed in certain malignancies. In the present study, the expression of MAC30 in a variety of normal and cancerous human gastrointestinal tissues, with special emphasis on pancreatic tissues was analyzed. Quantitative RT-PCR was utilized to compare MAC30 expression levels. In situ hybridization and immunohistochemistry were carried out to localize MAC30 mRNA and protein expression in normal and cancerous tissue samples of the esophagus, stomach, colon and pancreas. Furthermore, the effects of TGF-beta on the transcription of MAC30 mRNA were examined in pancreatic cancer cells. MAC30 mRNA was expressed in a wide variety of normal human tissues, being most abundant in testicular and gastric tissue samples. MAC30 mRNA levels were significantly increased in breast and colon cancer, but significantly decreased in pancreatic and renal cancer. TGF-beta down-regulated MAC30 mRNA levels in certain pancreatic cancer cells. MAC30 protein was localized in normal pancreatic tissues, mainly in acinar and islet cells, and in normal colon, gastric and esophageal tissues especially in the mucosal cells. MAC30 was strongly present in tubular complexes in pancreatic cancer tissues but weak to absent in pancreatic cancer cells of primary tumors and metastases. In contrast, esophageal, gastric and colon tumors displayed strong MAC30 immunoreactivity in the cancer cells. In conclusion, MAC30 is expressed in various normal and diseased human tissues. MAC30 up-regulation in certain tumors and down-regulation in others suggests that this protein plays a distinct role in human malignancies.

Paper title : Identification of cholesterol-regulating genes by targeted RNAi screening.

Doi : https://doi.org/10.1016/j.cmet.2009.05.009

Abstract : Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.

Paper title : A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes.

Doi : https://doi.org/10.1038/s41589-021-00907-2

Abstract : Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalog consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio- and stereoselective ligand for the protein transmembrane protein 97 (Tmem97/the σ2 receptor), enabling us to reconstruct the 20(S)-OHC-Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.

Paper title : Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97.

Doi : https://doi.org/10.3390/ijms24076381

Abstract : Sigma-2 receptor (S2R) is a S2R ligand-binding site historically associated with reportedly 21.5 kDa proteins that have been linked to several diseases, such as cancer, Alzheimer's disease, and schizophrenia. The S2R is highly expressed in various tumors, where it correlates with the proliferative status of the malignant cells. Recently, S2R was reported to be the transmembrane protein TMEM97. Prior to that, we had been investigating the translocator protein (TSPO) as a potential 21.5 kDa S2R candidate protein with reported heme and sterol associations. Here, we investigate the contributions of TMEM97 and TSPO to S2R activity in MCF7 breast adenocarcinoma and MIA PaCa-2 (MP) pancreatic carcinoma cells. Additionally, the role of the reported S2R-interacting partner PGRMC1 was also elucidated. Proximity ligation assays and co-immunoprecipitation show a functional association between S2R and TSPO. Moreover, a close physical colocalization of TMEM97 and TSPO was found in MP cells. In MCF7 cells, co-immunoprecipitation only occurred with TMEM97 but not with PGRMC1, which was further confirmed by confocal microscopy experiments. Treatment with the TMEM97 ligand 20-(S)-hydroxycholesterol reduced co-immunoprecipitation of both TMEM97 and PGRMC1 in immune pellets of immunoprecipitated TSPO in MP cells. To the best of our knowledge, this is the first suggestion of a (functional) interaction between TSPO and TMEM97 that can be affected by S2R ligands.

Paper title : Identification and characterization of genes differentially expressed in meningiomas.

Doi : https://doi.org/Not available

Abstract : Meningiomas are common tumours derived from the thin membrane that surrounds the brain and spinal cord. Currently, the molecular mechanisms responsible for the initiation and progression of these tumors are largely unknown. Toward the elucidation of such mechanisms, we have formulated an experimental design utilizing the technique of subtractive hybridization that is aimed at identifying the changes in gene expression between intracranial meningiomas and their normal precursor cells, leptomeningeal cells. We report here the identification and initial characterization of three genes whose expression is altered or aberrant in meningioma cell lines and tumours relative to cultures of normal leptomeningeal cells. Complementary DNA probes from one of these genes detect transcripts of altered size in several meningiomas relative to normal leptomeningeal cells. Another of these genes demonstrates decreased expression in meningiomas and in tumours associated with the disorder neurofibromatosis 2. A third gene isolated by this procedure is differentially expressed in both meningiomas and breast carcinomas. Therefore, the decreased expression of these genes may play roles in growth-regulatory pathways that are abrogated not only in meningiomas, but in other tumor types as well.

Paper title : S2R(Pgrmc1): the cytochrome-related sigma-2 receptor that regulates lipid and drug metabolism and hormone signaling.

Doi : https://doi.org/10.1517/17425255.2012.658367

Abstract : INTRODUCTION: S2R (sigma-2 receptor)/Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome-related protein that binds directly to heme and various pharmacological compounds. S2R(Pgrmc1) also associates with cytochrome P450 proteins, the EGFR receptor tyrosine kinase and the RNA-binding protein PAIR-BP1. S2R(Pgrmc1) is induced in multiple types of cancer, where it regulates tumor growth and is implicated in progesterone signaling. S2R(Pgrmc1) also increases cholesterol synthesis in non-cancerous cells and may have a role in modulating drug metabolizing P450 proteins. AREAS COVERED: This review covers the independent identification of S2R and Pgrmc1 and their induction in cancers, as well as the role of S2R(Pgrmc1) in increasing cholesterol metabolism and P450 activity. This article was formed through a PubMed literature search using, but not limited to, the terms sigma-2 receptor, Pgrmc1, Dap1, cholesterol and aromatase. EXPERT OPINION: Multiple laboratories have shown that S2R(Pgrmc1) associates with various P450 proteins and increases cholesterol synthesis via Cyp51. However, the lipogenic role of S2R(Pgrmc1) is tissue-specific. Furthermore, the role of S2R(Pgrmc1) in regulating P450 proteins other than Cyp51 appears to be highly selective, with modest inhibitory activity for Cyp3A4 in vitro and a complex regulatory pattern for Cyp21. Cyp19/aromatase is a therapeutic target in breast cancer, and S2R(Pgrmc1) activated Cyp19 significantly in vitro but modestly in biochemical assays. In summary, S2R(Pgrmc1) is a promising therapeutic target for cancer and possibly cholesterol synthesis but research to date has not identified a major role in P450-mediated drug metabolism.

Paper title : Structures of the σ₂ receptor enable docking for bioactive ligand discovery.

Doi : https://doi.org/10.1038/s41586-021-04175-x

Abstract : The σ₂ receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ₁ receptor. Crystal structures of two ligands bound to the σ₂ receptor confirmed the docked poses. To investigate the contribution of the σ₂ receptor in pain, two potent σ₂-selective ligands and one potent σ₁/σ₂ non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ₂ receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.

Paper title : Complete sequencing and characterization of 21,243 full-length human cDNAs.

Doi : https://doi.org/10.1038/ng1285

Abstract : As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Paper title : Sigma-2 receptor ligands: neurobiological effects.

Doi : https://doi.org/10.2174/0929867322666150114163607

Abstract : Sigma-2 receptor is a widely distributed protein, which can modulate cell proliferation and involved in the pathogenesis of tumor. Photoaffinity labelling techniques testified that its molecular size is about 18 kDa. Recent studies indicated that sigma-2 receptor modulates the cytosolic Ca2+ concentration, dopaminergic transmission, and cocaine-induced addiction behavior. Some sigma-2 receptor ligands (ditolylguanidine, afobazole, etc) display the neuroprotective effect. Although sigma-2 receptor hasn't been cloned, tens of sigma-2 receptor ligands, which demonstrate high affinity and selectivity, have been identified in the past decade. In this review, we mainly focus on these series of selective sigma-2 receptor ligands, their neuropsychological effects, and molecular probes for tracing sigma-2 receptors in central nervous system.

Paper title : Coordinate up-regulation of TMEM97 and cholesterol biosynthesis genes in normal ovarian surface epithelial cells treated with progesterone: implications for pathogenesis of ovarian cancer.

Doi : https://doi.org/10.1186/1471-2407-7-223

Abstract : BACKGROUND: Ovarian cancer (OvCa) most often derives from ovarian surface epithelial (OSE) cells. Several lines of evidence strongly suggest that increased exposure to progesterone (P4) protects women against developing OvCa. However, the underlying mechanisms of this protection are incompletely understood. METHODS: To determine downstream gene targets of P4, we established short term in vitro cultures of non-neoplastic OSE cells from six subjects, exposed the cells to P4 (10-6 M) for five days and performed transcriptional profiling with oligonucleotide microarrays containing over 22,000 transcripts. RESULTS: We identified concordant but modest gene expression changes in cholesterol/lipid homeostasis genes in three of six samples (responders), whereas the other three samples (non-responders) showed no expressional response to P4. The most up-regulated gene was TMEM97 which encodes a transmembrane protein of unknown function (MAC30). Analyses of outlier transcripts, whose expression levels changed most significantly upon P4 exposure, uncovered coordinate up-regulation of 14 cholesterol biosynthesis enzymes, insulin-induced gene 1, low density lipoprotein receptor, ABCG1, endothelial lipase, stearoyl- CoA and fatty acid desaturases, long-chain fatty-acyl elongase, and down-regulation of steroidogenic acute regulatory protein and ABCC6. Highly correlated tissue-specific expression patterns of TMEM97 and the cholesterol biosynthesis genes were confirmed by analysis of the GNF Atlas 2 universal gene expression database. Real-time quantitative RT-PCR analyses revealed 2.4-fold suppression of the TMEM97 gene expression in short-term cultures of OvCa relative to the normal OSE cells. CONCLUSION: These findings suggest that a co-regulated transcript network of cholesterol/lipid homeostasis genes and TMEM97 are downstream targets of P4 in normal OSE cells and that TMEM97 plays a role in cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in OSE cells might play a role in conferring protection against OvCa.

Paper title : N-terminome analysis of the human mitochondrial proteome.

Doi : https://doi.org/10.1002/pmic.201400617

Abstract : The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labelling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The doublet N-terminal oriented proteomics (dN-TOP) strategy based on a double light/heavy TMPP labelling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523 (http://proteomecentral.proteomexchange.org/dataset/PXD001521, http://proteomecentral.proteomexchange.org/dataset/PXD0001522 and http://proteomecentral.proteomexchange.org/dataset/PXD001523, respectively).

Paper title : Sigma-2 receptor ligands and their perspectives in cancer diagnosis and therapy.

Doi : https://doi.org/10.1002/med.21297

Abstract : The sigma-2 receptor is highly expressed in various rapidly proliferating cancer cells and regarded as a cancer cell biomarker. Selective sigma-2 ligands have been shown to specifically label the tumor sites, induce cancer cells to undergo apoptosis, and inhibit tumor growth. Sigma-2 ligands are potentially useful as cancer diagnostics, anticancer therapeutics, or adjuvant anticancer treatment agents. However, both the cloning of this receptor and the identification of its endogenous ligand have not been successful, and the lack of structural information has severely hindered the understanding of its physiological roles, its signaling pathways, and the development of more selective sigma-2 ligands. Recent data have implicated that sigma-2 binding sites are within the lipid rafts and that PGRMC1 (progesterone receptor membrane component 1) complex and sigma-2 receptor may be coupled with EGFR (epidermal growth factor receptor), mTOR (mammalian target of rapamycin), caspases, and ion channels. Due to its promising applications in cancer management, there are rapidly increasing research efforts that are being directed into this field. This review article updates the current understanding of sigma-2 receptor and its potential physiological roles, applications, interaction with other effectors, with special focuses on the development of sigma-2 ligands, their chemical structures, pharmacological profiles, applications in imaging and anticancer therapy.

Paper title : Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells.

Doi : https://doi.org/10.1093/hmg/ddw204

Abstract : Niemann-Pick type C disease (NP-C) is a progressive lysosomal lipid storage disease caused by mutations in the NPC1 and NPC2 genes. NPC1 is essential for transporting cholesterol and other lipids out of lysosomes, but little is known about the mechanisms that control its cellular abundance and localization. Here we show that a reduction of TMEM97, a cholesterol-responsive NPC1-binding protein, increases NPC1 levels in cells through a post-transcriptional mechanism. Reducing TMEM97 through RNA-interference reduces lysosomal lipid storage and restores cholesterol trafficking to the endoplasmic reticulum in cell models of NP-C. In TMEM97 knockdown cells, NPC1 levels can be reinstated with wild type TMEM97, but not TMEM97 missing an ER-retention signal suggesting that TMEM97 contributes to controlling the availability of NPC1 to the cell. Importantly, knockdown of TMEM97 also increases levels of residual NPC1 in NPC1-mutant patient fibroblasts and reduces cholesterol storage in an NPC1-dependent manner. Our findings propose TMEM97 inhibition as a novel strategy to increase residual NPC1 levels in cells and a potential therapeutic target for NP-C.

Paper title : Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

Doi : https://doi.org/10.1038/s41598-018-35430-3

Abstract : CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (σ2R) radioligands, [125I]RHM-4 or [3H]DTG, but a reduction in the receptor affinity of both radioligands was observed. TMEM97 KO resulted in a complete loss of binding of [125I]RHM-4 and a significant reduction in binding of [3H]DTG. TMEM97 KO and PGRMC1 KO resulted in an equal reduction in the rate of uptake of fluorescently-tagged or 3H-labeled LDL, and knocking out both proteins did not result in a further rate of reduction of LDL uptake. Confocal microscopy and Proximity Ligation Assay studies indicated a clear co-localization of LDLR, PGRMC1 and TMEM97. These data indicate that the formation of a ternary complex of LDLR-PGRMC1-TMEM97 is necessary for the rapid internalization of LDL by LDLR.