Type II Membrane Protein Research Articles

Evaluating the impact of the membrane thickness on the function of the intramembrane protease GlpG

Cellular membranes exhibit a huge diversity of lipids and membrane proteins that differ in their properties and chemical structure. Cells organize these molecules into distinct membrane compartments characterized by specific lipid profiles and hydrophobic thicknesses of the respective domains. If a hydrophobic mismatch occurs between a membrane protein and the surrounding lipids, there can be functional consequences such as reduced protein activity. This phenomenon has been extensively studied for single-pass transmembrane proteins, rhodopsin, and small polypeptides such as gramicidin. Here, we investigate the E. coli rhomboid intramembrane protease GlpG as a model to systematically explore the impact of membrane thickness on GlpG activity. We used fully saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) model lipids and altered membrane thickness by varying the cholesterol content. Physical membrane parameters were determined by 2H and 31P NMR spectroscopy and correlated with GlpG activity measurements in the respective host membranes. Differences in bulk and annular lipids as well as alterations in protein structure in the respective host membranes were determined using molecular dynamics simulations. Our findings indicate that GlpG can influence the membrane thickness in DLPC/cholesterol membranes but not in DMPC/cholesterol membranes. Moreover, we observe that GlpG protease activity is reduced in DLPC membranes at low cholesterol content, which was not observed for DMPC. While a change in GlpG activity can already be due to smallest differences in the lipid environment, potentially enabling allosteric regulation of intramembrane proteolysis, there is no overall correlation to cholesterol-mediated lipid bilayer organization and phase behavior. Additional factors such as the influence of cholesterol on membrane bending rigidity and curvature energy need to be considered. In conclusion, the functionality of α-helical membrane proteins such as GlpG relies not only on hydrophobic matching but also on other membrane properties, specific lipid interaction, and the composition of the annular layer.

Transmembrane Domains of Bitopic Proteins As a Key to Understand the Cellular Signaling

This work presents in a systematic manner key modeling results corroborated by experimental biophysical data and obtained by the authors during long-term research on bitopic (single-pass) membrane proteins (BMP), which are the crucial elements of cell signaling. The manuscript does not claim to be a comprehensive review on the topic, whereby the authors did not aim to describe accurately the current state of the art, given the numerous reliable publications. Rather, it is an essay illustrating the authors’ understanding of the basic principles in organization of transmembrane protein domains (TMD) and their contribution to the cell functioning. Among the key topics highlighted in the present work are the fine-tuned processes of TMD oligomerization and direct contribution of the dynamic membrane environment to this process, the key role of TMD in the functioning of cell receptors and mutual relations between all components of protein-membrane complexes during the signal transduction in normal and pathological conditions.

An extracellular vesicle delivery platform based on the PTTG1IP protein

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.

Weaker neuroligin 2 - neurexin 1β interaction tethers membranes and signal synaptogenesis through clustering.

Single-pass transmembrane proteins neuroligin (NL) and neurexin (NRX) constitute a pair of synaptic adhesion molecules (SAMs) that are essential for the formation of functional synapses. Binding affinities vary by ∼ 1000 folds between arrays of NL and NRX subtypes, which contribute to chemical and spatial specificities. Current structures are obtained with truncated extracellular domains of NL and NRX and are limited to the higher-affinity NL1/4-NRX complexes. How NL-NRX interaction leads to functional synapses remains unknown. Here we report structures of full-length NL2 alone, and in complex with NRX1β in several conformations, which has the lowest affinity among major NL-NRX subtypes. We show how conformational flexibilities may help in adapting local membrane geometry, and reveal mechanisms underlying variations in NL-NRX affinities modulation. We further show that, despite lower affinity, NL2-NRX1β interaction alone is capable of tethering different lipid membranes in total reconstitution, and that NL2 and NRX1β cluster at inter-cellular junctions without the need of other synaptic components. In addition, NL2 combines with the master post-synaptic scaffolding protein gephyrin and clusters neurotransmitter receptors at cellular membrane. These findings suggest dual roles of NL2 - NRX1β interaction - both as mechanical tether, and as signaling receptors, to ensure correct spatial and chemical coordination between two cells to generate function synapses.

Galvanin is an electric-field sensor for directed cell migration.

Directed cell migration is critical for the rapid response of immune cells, such as neutrophils, following tissue injury or infection. Endogenous electric fields, generated by the disruption of the transepithelial potential across the skin, help to guide the movement of immune and skin cells toward the wound site. However, the mechanisms by which cells sense these physical cues remain largely unknown. Through a CRISPR-based screen, we identified Galvanin, a previously uncharacterized single-pass transmembrane protein that is required for human neutrophils to change their direction of migration in response to an applied electric field. Our results indicate that Galvanin rapidly relocalizes to the anodal side of a cell on exposure to an electric field, and that the net charge on its extracellular domain is necessary and sufficient to drive this relocalization. The spatial pattern of neutrophil protrusion and retraction changes immediately upon Galvanin relocalization, suggesting that it acts as a direct sensor of the electric field that then transduces spatial information about a cell's electrical environment to the migratory apparatus. The apparent mechanism of cell steering by sensor relocalization represents a new paradigm for directed cell migration.

Susceptibility of BS90 Biomphalaria glabrata snails to infection by SmLE Schistosoma mansoni segregates as a dominant allele in a cluster of polymorphic genes for single-pass transmembrane proteins.

The trematodes that cause schistosomiasis in humans require aquatic snails as intermediate hosts. Identifying the genes in snails at which allelic variation controls resistance to infection by schistosomes could lead to novel ways to break the cycle of transmission. We therefore mapped genetic variation within the BS90 population of Biomphalaria glabrata snails that controls their resistance to infection by the SmLE population of Schistosoma mansoni. A marker in the PTC2 genomic region strongly associates with variation in resistance. The S-haplotype, which confers increased susceptibility, appears to be almost completely dominant to the R-haplotype, which confers increased resistance. This result suggests a model in which the parasite must match a molecule on the host side to successfully infect. The genomic region surrounding our marker shows high structural and sequence variability between haplotypes. It is also highly enriched for genes that code for single-pass transmembrane (TM1) genes. Several of the TM1 genes present on the S-haplotype lack orthologs on the R-haplotype, which makes them intriguing candidate genes in a model of dominant susceptibility. These results add to a growing body of work that suggests TM1 genes, especially those in this exceptionally diverse genomic region, may play an important role in snail-schistosome compatibility polymorphisms.

SARS-CoV-2 envelope protein alters calcium signaling via SERCA interactions

The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.

ELFN1 is a new extracellular matrix (ECM)-associated protein

AimsThe ELFN1, discovered in 2007, is a single-pass transmembrane protein. Studies conducted thus far to elucidate the function of the Elfn1 have been limited only to animal studies. These studies have reported that ELFN1 is a universal binding partner of metabotropic glutamate receptors (mGluRs) in the central nervous system and its functional deficiency has been associated with the pathogenesis of neurological and neuropsychiatric diseases. In 2021, we described the first disease-associated human ELFN1 pathogenic gene mutation. Severe joint laxity, which was the most striking finding of this new disease and was clearly seen in the patients since early infancy, showed that the ELFN1 may have a possible function in the connective tissue besides the nervous system. Here, we present the first experimental evidence of the extracellular matrix (ECM)-related function of the ELFN1. Materials and methodsPrimary skin fibroblasts were isolated from the skin biopsies of ELFN1 mutated patients and healthy foreskin donors. For the clinical trial in a dish, in vitro ECM and DEM (decellularized ECM) models were created from skin fibroblasts. All the in vitro models were comparatively characterized and analyzed. Key findingsThe mutation in the ELFN1 signal peptide region of patients resulted in a severe lack of ELFN1 expression and dramatically altered the characteristic morphology and behavior (growth, proliferation, and motility) of fibroblasts. SignificanceWe propose that ELFN1 is involved in the cell-ECM attachment, and its deficiency is critical enough to cause a loss of cell motility and soft ECM stiffness.

Conformations of influenza A M2 protein in DOPC/DOPS and E. coli native lipids and proteins

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IM2) protein reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPS) bilayers to those in isolated Escherichia coli (E. coli) membranes, having preserved its native proteins and lipids. IM2 is a single-pass transmembrane protein known to assemble into a homo-tetrameric proton channel. To represent this channel, we made a construct containing the IM2’s TMD region flanked by the juxtamembrane residues. The single cysteine substitution, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the electron spin resonance (ESR) study. For this particular residue, we probed the conformations of the spin-labeled IM2 reconstituted in DOPC/DOPS and isolated E. coli membranes using continuous-wave ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The continuous-wave ESR spectra corresponded to very slow spin-label motion in both environments. In all cases, the DEER data were reconstructed into distance distributions with well-resolved peaks at 1.68 and 2.37 nm in distance and amplitude ratios of 1.41 ± 0.2 and 2:1, respectively. This suggests four nitroxide spin labels located at the corners of a square, indicative of an axially symmetric tetramer. The distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IM2 TMD tetramer in agreement with the model. Thus, we can conclude that, under conditions of pH 7.4 used in this study, IM2 TMD has similar conformations in model lipid bilayers and membranes made of native E. coli lipids and proteins of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Essential role of N-terminal SAM regions in STIM1 multimerization and function

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Recombinant Klotho protein protects pulmonary alveolar epithelial cells against sepsis-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway.

Inflammation-induced apoptosis of alveolar type II epithelial cells is a primary contributor to sepsis-induced acute respiratory distress syndrome (ARDS). Klotho is a single-pass transmembrane protein with anti-inflammatory and anti-apoptotic effects. However, the role and mechanism of Klotho in the development of ARDS remains unknown. This study aimed to investigate the effect of Klotho on sepsis-induced apoptosis in human pulmonary alveolar epithelial cells (HPAEpiCs) together with the potential mechanism. Cecal ligation and puncture (CLP) were performed to generate an in vivo sepsis model, and HPAEpiCs were treated with lipopolysaccharide (LPS) to mimic sepsis in vitro. Both models were administered recombinant Klotho protein. The morphology of the lung tissue was observed, and apoptotic cells and cell viability were detected. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA), while the expression of Bcl-2, Bax and cleaved caspase-3 was detected with western blotting. Klotho reversed the CLP-induced decrease in mouse survival in vivo (p < 0.001) and increased inflammatory cell infiltration and inflammatory substance exudation in the lung tissue of mice with sepsis (both p < 0.001). Klotho also suppressed apoptosis (p < 0.001) as demonstrated by IL-1β, IL-6 and TNF-α expression (all p < 0.001), and Bcl-2/Bax/caspase-3 pathway activation (p < 0.001). Klotho pretreatment significantly prevented LPS-induced apoptosis in vitro (p < 0.001), as demonstrated by IL-1β, IL-6 and TNF-α upregulation (all p < 0.001); and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs (p < 0.001). This study demonstrated that Klotho can ameliorate acute lung injury (ALI) induced by sepsis by inhibiting inflammatory responses and exerting anti-apoptotic effects by suppressing Bcl-2/Bax/caspase-3 pathway activation.

Transmembrane signaling through single-spanning receptors modulated by phase separation at the cell surface

A wide variety of transmembrane signals are transduced by cell-surface receptors that activate intracellular signaling molecules. In particular, receptor clustering in the plasma membrane plays a critical role in these processes. Single-spanning or single-pass transmembrane proteins are among the most significant types of membrane receptors, which include adhesion receptors, such as integrins, CD44, cadherins, and receptor tyrosine kinases. Elucidating the molecular mechanisms underlying the regulation of the activity of these receptors is of great significance. Liquid–liquid phase separation (LLPS) is a recently emerging paradigm in cellular physiology for the ubiquitous regulation of the spatiotemporal dynamics of various signaling pathways. This study describes the emerging features of transmembrane signaling through single-spanning receptors from the perspective of phase separation. Possible physicochemical modulations of LLPS-based transmembrane signaling are also discussed.

Abstract 1900: TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer

Abstract Cadherin 17 (CDH17) is a cell-to-cell adhesion protein that is a member of the cadherin superfamily. In normal tissues, expression of this single pass transmembrane protein is restricted to the lateral surfaces of intestinal and pancreatic ductal epithelial cells. However, in cancer, CDH17 is frequently overexpressed in tumors of the colon, stomach, and pancreas. The selective expression of this cell surface protein in these hard-to-treat cancer makes it an attractive target for the development of antibody-based therapeutics. Here, we describe the preclinical development of TORL-3-600, a novel CDH17-targeting antibody drug conjugate (ADC). CDH17 specific monoclonal antibodies were generated using traditional hybridoma technology and splenocytes isolated from mice immunized with cocktails of NIH3T3 cells overexpressing full length hCHD17 plus purified mammalian expressed hCDH17 extracellular domain protein (aa 23-787). Selective mAb binding was confirmed by flow cytometry and mAb internalization rate was assessed by immunofluorescence (IF). TORL-3-600 was generated from a fully humanized CDH17 mAb by MMAE conjugation with a cleavable linker. Cell membrane staining of CDH17 in patient tumor microarrays (TMAs), cell line (CDX) and patient derived xenografts (PDX) was evaluated by IHC assay. Selective binding of the TORL-3-600 to CDH17 was confirmed in human cancer cell lines and cells engineered to overexpress CDH17. Binding of TORL-3-600 to cell surface CDH17 induced Internalization and translocation to the lysosome of the protein-ADC complex for release of the MMAE payload. Treatment with TORL-3-600 induced significant regressions and tumor growth inhibition (TGI) in four CDH17-positive (CDH17+) human colorectal cancer (CRC) CDXs (103.6 - 140.4% TGI) and three CDH17+ CRC PDXs (63.3 - 102.2% TGI). Responses in these models were sustained for up to nine-weeks following cessation of treatment. Sustained inhibition of xenograft tumor progression was also observed in a cell line model of CDH17+ pancreatic cancer (85.8% TGI). In contrast, greatly reduced responses (37.3 - 58.2% TGI) to TORL-3-600 were observed in the CDH17- human colon cancer CDX and PDX models. Each of the doses tested in this study were well tolerated in mice with no dose-limiting toxicities observed. Analyses of large human patient TMAs by CDH17 IHC assay demonstrated detectable expression of CDH17 in 90.1% (173/193) of CRC, 51.8% (86/166) of gastric and 20.4% (69/331) of pancreatic cancers. These numbers suggest that an ADC directed against CDH17 could provide benefit to a significant number of patients diagnosed with these cancers. These data support the clinical development of TORL-3-600 for the treatment of CDH17+ cancers. TORL-3-600 has completed IND enabling toxicity studies with acceptable PK and toxicity profiles and is now in phase 1 clinical testing (NCT05948826). Citation Format: Neil A. O'Brien, Martina SJ McDermott, Jun Zhang, Ming Lu, Ke Wei Gong, Benjamin Hoffstrom, Wei Ping Jia, Tong Luo, Athena M. Madrid, Min Liang, John A. Glaspy, Dennis J. Slamon. TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1900.

The Small Interactor of PKD2 protein promotes the assembly and ciliary entry of the Chlamydomonas PKD2-mastigoneme complexes.

In Chlamydomonas, the channel polycystin 2 (PKD2) is primarily present in the distal region of cilia, where it is attached to the axoneme and mastigonemes, extracellular polymers of MST1. In a smaller proximal ciliary region that lacks mastigonemes, PKD2 is more mobile. We show that the PKD2 regions are established early during ciliogenesis and increase proportionally in length as cilia elongate. In chimeric zygotes, tagged PKD2 rapidly entered the proximal region of PKD2-deficient cilia, whereas the assembly of the distal region was hindered, suggesting that axonemal binding of PKD2 requires de novo assembly of cilia. We identified the protein Small Interactor of PKD2 (SIP), a PKD2-related, single-pass transmembrane protein, as part of the PKD2-mastigoneme complex. In sip mutants, stability and proteolytic processing of PKD2 in the cell body were reduced and PKD2-mastigoneme complexes were absent from the cilia. Like the pkd2 and mst1 mutants, sip mutant cells swam with reduced velocity. Cilia of the pkd2 mutant beat with an increased frequency but were less efficient in moving the cells, suggesting a structural role for the PKD2-SIP-mastigoneme complex in increasing the effective surface of Chlamydomonas cilia.

Optimizing Transmembrane Protein Assemblies in Nanodiscs for Structural Studies: A Comprehensive Manual.

Membrane protein structures offer a more accurate basis for understanding their functional correlates when derived from full-length proteins in their native lipid environment. Producing such samples has been a primary challenge in the field. Here, we present robust, step-by-step biochemical and biophysical protocols for generating monodisperse assemblies of full-length transmembrane proteins within lipidic environments. These protocols are particularly tailored for cases where the size and molecular weight of the proteins align closely with those of the lipid islands (nanodiscs). While designed for single-span bitopic membrane proteins, these protocols can be easily extended to proteins with multiple transmembrane domains. The insights presented have broad implications across diverse fields, including biophysics, structural biology, and cryogenic electron microscopy (cryo-EM) studies. Key features • Overview of the sample preparation steps from protein expression and purification and reconstitution of membrane proteins in nanodiscs, as well as biobeads and lipids preparation. • Focus on single-span bitopic transmembrane proteins. • Includes protocols for validation procedures via characterization using biochemical, biophysical, and computational techniques. • Guide for cryogenic electron microscopy data acquisition from vitrification to image processing.

Probing the formation of a hetero-dimeric membrane transport complex with dual in vitro and in silico mutagenesis.

The reversible association of transmembrane helices is a fundamental mechanism in how living cells convey information and respond to physiological events. The cardiac calcium transport regulator phospholamban (PLN) is an example of a single-span transmembrane protein that populates a variety of reversible and competing oligomeric states. PLN primarily forms monomers and pentamers in the membrane, where the PLN pentamer is a storage form and the PLN monomer forms a hetero-dimeric inhibitory complex with SERCA. The binding affinity and free-energy of formation of the SERCA-PLN complex in a membrane have not been determined. As is the case for most transmembrane protein interactions, measuring these quantities experimentally is extremely challenging. In this study, we estimated binding affinities by employing in silico alchemical free-energy calculations for all PLN transmembrane alanine substitutions in a membrane bilayer. The binding affinities were calculated separately for the SERCA-PLN complex, a PLN monomer, and a PLN pentamer and compared to in vitro functional measurements of SERCA regulation by the PLN alanine substitutions. Initially, the changes in SERCA inhibition by PLN alanine substitutions were compared to the changes in free energy for the SERCA-PLN complex formed from the PLN monomer. However, the functional data for the PLN alanine substitutions were better explained by the formation of the SERCA-PLN complex directly from the PLN pentamer. This finding points to an inhibitory mechanism favoring conformational selection of SERCA and the interaction of a PLN pentamer with SERCA for 'delivery' of a PLN monomer to the inhibitory site. The implications of these findings suggest that the energetics of helix exchange between homo- and hetero-oligomeric signaling complexes is favored over an intermediate involving a free monomeric helix in the membrane bilayer.

Abstract B133: Anti-tumor efficacy of GMF-1A3-MMAE, an antibody drug conjugate targeting cell surface cleaved Amphiregulin in endocrine-resistant breast cancer

Abstract The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we describe the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. A pan-cancer tissue microarray analysis indicates that the target is commonly detected at high levels (&amp;gt; 50% of cases) in breast, prostate, liver and lung cancer. In normal tissue microarrays, cleaved Amphiregulin was generally detected at moderate levels in several tissues with only 7 of 93 tissue cores having levels comparable to the high levels commonly found in tumors. These data suggest therapeutic opportunities for GMF-1A3-based ADCs in multiple malignancies, however the broad off-tumor detection of cleaved Amphiregulin suggest linker/payload optimization may be necessary for an adequate therapeutic window. Citation Format: Kristopher A Lofgren, Paraic A Kenny. Anti-tumor efficacy of GMF-1A3-MMAE, an antibody drug conjugate targeting cell surface cleaved Amphiregulin in endocrine-resistant breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B133.

NXPE2 Is the Target of Ter-119 When Complexed with Gypa in Mice

Background: Red blood cells (RBCs) have a complex biology that allows regulation of metabolism, cellular rigidity, ion exchange, pH, and molecular communication with the vasculature. A central regulator of these biologies are macrocomplexes of integral membrane proteins that are both linked to cytoskeletal proteins and also differentially bind to metabolic enzymes based upon oxygenation status of the RBC. RBC surface staining, co-immunoprecipitation, and cross-linking proteomic approaches have generated a model of the macrocomplex encompassing Band 3, Rh, RhAG, CD47, glycophorin A, glycophorin B, and LW. TER-119 is a monoclonal antibody with exquisite specificity for the erythroid lineage in mice. However, the molecular target of TER-119 has remained unknown. TER-119 immunoprecipitates 4 specific protein bands, two of which are glycophorin A (GYPA) monomers or homodimers respectively; however, TER-119 is non-reactive to GYPA when assayed through Western blots and flow cytometry with erythroid cell lines expressing GYPA. These data led the originators of TER-119 to conclude it binds to a GYPA associated protein, but not GYPA itself. In contrast, others have concluded that TER-119 binds GYPA directly, and have inferred GYPA biology based upon TER-119 reactivity. Methods: TER-119 reactivity with peripheral blood RBCs from mice or from transfected HEK cells was determined by flow cytometry. Mice were genotyped using a dense array of 143,000 markers. Quantitative trait loci (QTL) analysis was carried out using TER-119 staining as a trait. cDNA for the open reading frames of the variants of interest in candidate genes or GYPA were cloned into eukaryotic expression vectors drive by the CMV promoter. HEK cells were transfected with expression vectors, in combination with a GFP expression plasmid. Cells were stained with TER-119 48 hours post transfection and analyzed by flow cytometry, gating on GFP positive cells to isolate the transfected population. Hematopoietic lineage expression of mRNA was obtained from the BloodSpot database. Results: The Diversity Outbred mice (DO mice) are an outbred population descended from 8 parental strains (A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). While analyzing RBC biology phenotypes in DO mice, using TER-119 as a marker of RBCs, we serendipitously observed TER-119 non-reactivity in peripheral blood of 5 out of 550 DO mice. QTL analysis using TER-119 reactivity as a trait identified a highly significant QTL (genome-wide adjusted p-value &amp;lt;1.11e-16) driven by alleles specific to WSB/EiJ mice. Analysis of each of the parental strains confirmed that only peripheral blood from WSB/EiJ mice was non-reactive with TER-119. The QTL region contained 4 genes with missense mutations specific to the WSB/EiJ strain (HTR3a, HTR3b, NXPE2, and NXPE4). TER-119 was weakly reactive with HEK cells transfected with NXPE2 (but not the other genes). However, TER-119 became strongly reactive with HEK cells co-transfected with NXPE2 and GYPA (but not the other genes co-transfected with GYPA). There is no GYPA coding variation present in 8 DO parental strains. NXPE2 mRNA is highly expressed in the murine erythroid lineage with lower expression in B cells and granulocytes. Discussion: These data demonstrate that TER-119 reactivity is conferred by co-expression of NXPE2 and GYPA. NXPE2 is a single pass transmembrane protein and the weak reactivity of TER-119 with NXPE2 alone suggests that TER-119 recognizes an epitope on NXPE2 that is allosterically enhanced by complexing with GYPA. However, other binding scenarios cannot be ruled out. These data are important for three reasons. First, they identify NXPE2 as a hitherto unrecognized surface protein specific to the erythroid lineage in mice. Second, they raise the possibility that the models of the macrocomplex are incomplete. Third, these findings may allow re-interpretation of existing data that assumed TER-119 recognized GYPA. Additional studies are required to determine what function (if any) NXPE2 plays in erythropoiesis in mice and whether this function translates to human NXPE2 or a homologous gene.

The role of structural heterogeneity in the homodimerization of transmembrane proteins.

The equilibrium association of transmembrane proteins plays a fundamental role in membrane protein function and cellular signaling. While the study of the equilibrium binding of single pass transmembrane proteins has received significant attention in experiment and simulation, the accurate assessment of equilibrium association constants remains a challenge to experiment and simulation. In experiment, there remain wide variations in association constants derived from experimental studies of the most widely studied transmembrane proteins. In simulation, state-of-the art methods have failed to adequately sample the thermodynamically relevant structures of the dimer state ensembles using coarse-grained models. In addition, all-atom force fields often fail to accurately assess the relative free energies of the dimer and monomer states. Given the importance of this fundamental biophysical process, it is essential to address these shortcomings. In this work, we establish an effective computational protocol for the calculation of equilibrium association constants for transmembrane homodimer formation. A set of transmembrane protein homodimers, used in the parameterization of the MARTINI v3 force field, are simulated using metadynamics, based on three collective variables. The method is found to be accurate and computationally efficient, providing a standard to be used in the future simulation studies using coarse-grained or all-atom models.

Serum Klotho Level and its Related Factors Among Male Opioids Addicts With Normal Renal Function Compared to Healthy Male Non-smokers and Smokers in Tabriz, Iran

Background: Klotho is an aging-suppressor gene that encodes a single-pass transmembrane protein and acts as a hormone. In this study, we aim to investigate the serum α-Klotho level in male opioids addicts with normal kidney function compared to healthy male non-smokers and smokers in Tabriz, Iran. Methods: Participnts were 87 men with normal kidney function referred to Sina Educational Research and Treatment Center in Tabriz, Iran (29 opioids addicts, 29 healthy non-smokers, and 29 healthy smokers). Blood samples were collected to measure the soluble a-Klotho level using an ELISA kit. Furthermore, blood creatinine (Cr) and hemoglobin (Hb) levels was measured. Body mass index (BMI) was also calculated for all participants. Results: In addicts, BMI, Hb, and Cr levels were significantly lower than in healthy non-smokers and smokers, but their Klotho level was higher (P&gt;0.05). The Klotho level in healthy smokers was significantly lower than in healthy non-smokers and addicts. The Klotho level of healthy smokers decreased as the pack year increased, but the duration of opioid addiction had no significant association with the Klotho level. There was no significant difference in the Klotho level between control groups (non-smokers and smokers) and men with addiction to different types of opioids. Conclusion: The Klotho level in male opioid addicts is significantly higher than in smokers. There is a significant negative correlation between BMI and Klotho levels among men with normal BMI and overweight. Further studies are recommended in these fields.