Multipass Transmembrane Protein Research Articles

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum

Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multi-pass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we employ high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system, with potential implications for therapeutic applications.

Methods for Engineering Binders to Multi-Pass Membrane Proteins.

Numerous potential drug targets, including G-protein-coupled receptors and ion channel proteins, reside on the cell surface as multi-pass membrane proteins. Unfortunately, despite advances in engineering technologies, engineering biologics against multi-pass membrane proteins remains a formidable task. In this review, we focus on the different methods used to prepare/present multi-pass transmembrane proteins for engineering target-specific biologics such as antibodies, nanobodies and synthetic scaffold proteins. The engineered biologics exhibit high specificity and affinity, and have broad applications as therapeutics, probes for cell staining and chaperones for promoting protein crystallization. We primarily cover publications on this topic from the past 10 years, with a focus on the different formats of multi-pass transmembrane proteins. Finally, the remaining challenges facing this field and new technologies developed to overcome a number of obstacles are discussed.

Small proteins modulate ion-channel-like ACD6 to regulate immunity in Arabidopsis thaliana

The multi-pass transmembrane protein ACCELERATED CELL DEATH 6 (ACD6) is an immune regulator in Arabidopsis thaliana with an unclear biochemical mode of action. We have identified two loci, MODULATOR OF HYPERACTIVE ACD6 1 (MHA1) and its paralog MHA1-LIKE (MHA1L), that code for ∼7kDa proteins, which differentially interact with specific ACD6 variants. MHA1L enhances the accumulation of an ACD6 complex, thereby increasing the activity of the ACD6 standard allele for regulating plant growth and defenses. The intracellular ankyrin repeats of ACD6 are structurally similar to those found in mammalian ion channels. Several lines of evidence link increased ACD6 activity to enhanced calcium influx, with MHA1L as a direct regulator of ACD6, indicating that peptide-regulated ion channels are not restricted to animals.

Current drugs for HIV-1: from challenges to potential in HIV/AIDS

The human immunodeficiency virus (HIV) persists in latently infected CD4+T cells and integrates with the host genome until cell death. Acquired immunodeficiency syndrome (AIDS) is associated with HIV-1. Possibly, treating HIV/AIDS is an essential but challenging clinical goal. This review provides a detailed account of the types and mechanisms of monotherapy and combination therapy against HIV-1 and describes nanoparticle and hydrogel delivery systems. In particular, the recently developed capsid inhibitor (Lenacapavir) and the Ainuovirine/tenofovir disoproxil fumarate/lamivudine combination (ACC008) are described. It is interestingly to note that the lack of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and the multipass transmembrane proteins serine incorporator 5 (SERINC5) may be one of the reasons for the enhanced infectivity of HIV-1. This discovery of SERINC3 and SERINC5 provides new ideas for HIV-1 medication development. Therefore, we believe that in treating AIDS, antiviral medications should be rationally selected for pre-exposure and post-exposure prophylaxis to avoid the emergence of drug resistance. Attention should be paid to the research and development of new drugs to predict HIV mutations as accurately as possible and to develop immune antibodies to provide multiple guarantees for the cure of AIDS.

SARS-CoV-2 nonstructural protein 6 from Alpha to Omicron: evolution of a transmembrane protein.

We recently reported that mutations in both the spike glycoprotein and nonstructural protein 6 (nsp6) were associated with attenuation of the SARS-CoV-2 Omicron BA.1 variant. While mutations in spike allow evasion of neutralizing antibodies and promote specific modes of viral entry, the role of nsp6 mutations in pathogenesis is less clear. Nsp6 is essential for modifying the endoplasmic reticulum and generating double-membrane vesicles, the site of viral RNA replication. To investigate the evolution of nsp6, we evaluated 91,596 high-confidence human SARS-CoV-2 whole-genome sequences across 19 variants and lineages. While nsp6 of early variants of concern, such as Alpha, Beta, and Gamma, carried a triple amino acid deletion (106-108, termed ΔSGF), the Delta, Epsilon, and Mu lineages retained the ancestral nsp6 sequence. For nsp6 in the emerging Omicron variants, we report a transition from an amino acid 105-107 ΔLSG deletion in BA.1 to increased dominance of the ΔSGF in BA.2 and subsequent lineages. Our findings indicate that deletion within nsp6 was independently selected in multiple lineages of SARS-CoV-2, both early and late in the pandemic. Analysis of SARS-CoV-2-related coronaviruses in bats and pangolins revealed nsp6 sequences similar to the ancestral SARS-CoV-2 virus, indicating that the deletion in nsp6 may be an adaptation to replication in humans. Analysis of nsp6 sequences from multiple coronaviruses predicts a multipass transmembrane protein with a conserved C-terminal domain. Monitoring and evaluating changes in nsp6 and other nonstructural proteins will contribute to our understanding of factors associated with the attenuation of pandemic coronaviruses. IMPORTANCE There is an ongoing need to evaluate genetic changes in SARS-CoV-2 for effects on transmission and pathogenesis. We recently reported an unexpected role for replicase component nsp6, in addition to changes in spike, in the attenuation of Omicron BA.1. In this commentary, we document a triple-amino-acid deletion in a predicted lumenal domain of nsp6 that was found in multiple, independent variants of SARS-CoV-2, including all recent Omicron lineages. Furthermore, we modeled the predicted structure of nsp6, implicating a multipass transmembrane architecture as conserved in members of the Coronaviridae family. This information can guide future studies investigating the role of nsp6 in the pathogenesis of existing and emerging coronaviruses.

Spinocerebellar ataxia 38: structure–function analysis shows ELOVL5 G230V is proteotoxic, conformationally altered and a mutational hotspot

Fatty acid elongase ELOVL5 is part of a protein family of multipass transmembrane proteins that reside in the endoplasmic reticulum where they regulate long-chain fatty acid elongation. A missense variant (c.689G>T p.Gly230Val) in ELOVL5 causes Spinocerebellar Ataxia subtype 38 (SCA38), a neurodegenerative disorder characterized by autosomal dominant inheritance, cerebellar Purkinje cell demise and adult-onset ataxia. Having previously showed aberrant accumulation of p.G230V in the Golgi complex, here we further investigated the pathogenic mechanisms triggered by p.G230V, integrating functional studies with bioinformatic analyses of protein sequence and structure. Biochemical analysis showed that p.G230V enzymatic activity was normal. In contrast, SCA38-derived fibroblasts showed reduced expression of ELOVL5, Golgi complex enlargement and increased proteasomal degradation with respect to controls. By heterologous overexpression, p.G230V was significantly more active than wild-type ELOVL5 in triggering the unfolded protein response and in decreasing viability in mouse cortical neurons. By homology modelling, we generated native and p.G230V protein structures whose superposition revealed a shift in Loop 6 in p.G230V that altered a highly conserved intramolecular disulphide bond. The conformation of this bond, connecting Loop 2 and Loop 6, appears to be elongase-specific. Alteration of this intramolecular interaction was also observed when comparing wild-type ELOVL4 and the p.W246G variant which causes SCA34. We demonstrate by sequence and structure analyses that ELOVL5 p.G230V and ELOVL4 p.W246G are position-equivalent missense variants. We conclude that SCA38 is a conformational disease and propose combined loss of function by mislocalization and gain of toxic function by ER/Golgi stress as early events in SCA38 pathogenesis.

Abstract 5320: The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets

Abstract In recent years, an increasing number of therapeutic antibodies have shown to be effective for the treatment of cancer and other diseases. However, limitations in the traditional discovery process, including immune tolerance of highly homologous genes, challenges with antibody sequence humanization, clone selection, and model selection for drug efficacy and safety evaluation, often hinder the process of identifying new therapeutic antibodies. The RenMice™ HiTS (Hyperimmune Target Specific) Platform is a library of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific drug target gene knocked out. These mice are designed to establish robust immune responses and generate antibodies that bind to more epitopes of the target protein, including conserved domains. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins (e.g. GPCRs/ion channels). Here, we show that the platform can be used to generate antibodies that cross-react with multiple species, like human, monkey, dog, and mouse targets, by immunizing with both human and mouse or dog antigen. We provide examples for newer campaigns, including species cross-reactivity and internalization of novel antibodies targeting NECTIN-4, and high-throughput in vivo efficacy screening of novel anti-PD-1 antibodies in wild-type mice. In the future, we will evaluate the preliminary toxicity of these cross-reactive antibodies in preclinical animal models. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of developable antibodies that recognize novel epitopes and challenging targets. Citation Format: Xiaoqian Zhang, Shufang Fu, Shujin Zhang, Xin Ji, Li Hui, James Jin, Jing Zhang. The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5320.

Abstract 1383: Mechanisms of ATG9 mediated aggrephagy initiation

Abstract Highly biosynthetic cancer cells produce proteins at a rate that may exceed the capacity of protein folding machinery, resulting in an accumulation of misfolded proteins that are poly-ubiquitinated and clustered into phase-separated ‘ubiquitin-rich’ condensates. These condensates are the initiation point for a form of autophagy called aggrephagy, which maintains cancer cell homeostasis by degrading the misfolded protein condensates via the lysosome. Aggrephagy is a cancer cell vulnerability, yet its mechanisms are poorly understood, particularly the mechanisms by which autophagy machinery recognizes the ubiquitin-rich condensates to initiate aggrephagy. Among 15 core autophagy proteins, ATG9A is the only multi-pass transmembrane protein and is essential for all known forms of autophagy. Recent evidence indicates that ATG9A is a lipid scramblase, in complex with ATG2A, that channels lipids to a growing autophagosome, which ultimately engulfs the ubiquitin-rich condensate. However, the mechanism of ATG9A recruitment to these condensates is not understood. Our data indicate that genetic or chemical manipulations of cells that cause ubiquitin-rich condensates to accumulate causing a corresponding accumulation of ATG9A. Furthermore, the induction of artificial ubiquitin-rich condensates, composed of chains of M1-linked ubiquitin, is sufficient to recruit ATG9A. Using a CRISPR knockout and reconstitution approach, we show that ATG9A is essential for assembling other autophagy machinery at ubiquitin-rich condensates. Together, our data support a model in which ATG9A recruitment is the initiating step for aggrephagy. Furthermore, our data suggest a signal or molecular pattern within the condensate promotes ATG9A recruitment to initiate aggrephagy. Our current work focuses on identifying features of ATG9A and components of the ubiquitin-rich condensate that cooperate to recruit ATG9A to these sites of aggrephagy initiation. Citation Format: Deshan Madhusanka, Colton McEwan, David Broadbent, Jens Schmidt, Joshua Andersen. Mechanisms of ATG9 mediated aggrephagy initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1383.

Discovery of antibodies targeting multipass transmembrane proteins using a suspension cell-based evolutionary approach

Due to their critical functions in cell sensing and signal processing, membrane proteins are highly preferred as pharmacological targets, and antibody drugs constitute the fastest growing category of therapeutic agents on the pharmaceutical market. However, major limitations exist in developing antibodies that recognize complex, multipass transmembrane proteins, such as G-protein-coupled receptors (GPCRs). These challenges, largely due to difficulties with recombinant expression of multipass transmembrane proteins, can be overcome using whole-cell screening techniques, which enable presentation of the functional antigen in its native conformation. Here, we developed suspension cell-based whole-cell panning methodologies to screen forspecific binders against GPCRs within a naive yeast-displayed antibody library. We implemented our strategy to discover high-affinity antibodies against four distinct GPCR target proteins, demonstrating the potential for our cell-based screening workflow to advance the discovery of antibody therapeutics targeting membrane proteins.

Copolymeric nanodiscs do not retain the native membrane environment, but do maintain protein-lipid interactions.

Polymer stabilized lipid-protein nanoparticles have become invaluable tools in membrane biology since they can isolate individual proteins with their surrounding lipids. These nanoparticles, termed nanodiscs, have historically relied on membrane scaffolding proteins, but can now be formed using amphipathic copolymers that directly intercalate into membranes. Since copolymeric nanodiscs are formed directly from membranes without requiring detergent solubilization, they have been presumed to retain the native membrane environment, but this assumption has not been thoroughly tested. To test if native membrane lipid packing is retained in styrene-maleic acid (SMA) and diisobutylene-maleic acid (DIBMA) stabilized nanodiscs, lipid packing was analyzed using solvatochromic dyes Laurdan, C-Laurdan, and Pro12A. SMA and DIBMA copolymeric nanodiscs were formed from membranes increasing in complexity from single and binary lipid mixture, to biomimetic ternary mixtures composed of palmitoyl-sphingomyelin (PSM), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and cholesterol, and ultimately to mammalian cellular membranes. For all synthetic model membranes, lipid packing of fluid-disordered membranes was significantly increased in both SMA and DIBMA nanodiscs compared to intact membranes, with DIBMA being the least perturbing. In contrast, lipid packing in ordered membranes was unaffected. For cellular membranes, lipid packing was also higher inside nanodiscs than in native membranes. Our data challenges the assumption that native membrane properties are retained inside nanodiscs. While not maintaining native membrane properties, nanodiscs can be useful models for studying protein-lipid interactions, as they are composed of membrane lipids. Interestingly, nanodiscs can exchange lipids through collisions, allowing tuning of their lipid compositions. We used this capability to analyze the preferred surrounding lipidome of multi-pass transmembrane proteins. Altogether, our studies demonstrate that copolymeric nanodiscs have different properties to those of intact membranes, but they are useful models in studying the paralipidomes of membrane proteins.

Visualization of translation and protein biogenesis at the ER membrane

The dynamic ribosome–translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3–7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation invitro and disrupted cortical histogenesis invivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Clptm1, a new target in suppressing epileptic seizure by regulating GABAA R-mediated inhibitory synaptic transmission in a PTZ-induced epilepsy model.

Disruption of gamma-amino butyric acid type A receptors (GABAA Rs) synaptic clustering and a decrease in the number of GABAA Rs in the plasma membrane are thought to contribute to alteration of the balance between excitatory and inhibitory neurotransmission, which promotes seizure induction and propagation. The multipass transmembrane protein cleft lip and palate transmembrane protein 1 (Clptm1) controls the forward trafficking of GABAA R, thus decaying miniature inhibitory postsynaptic current (mIPSC) of inhibitory synapses. In this study, using a pentylenetetrazol (PTZ)-induced epilepsy rat model, we found that Clptm1 regulates epileptic seizures by modulating GABAA R-mediated inhibitory synaptic transmission. First, we showed that Clptm1 expression was elevated in the PTZ-induced epileptic rats. Subsequently, we found that downregulation of Clptm1 expression protected against PTZ-induced seizures, which was attributed to an increase in the number of GABAA Rγ2s in the plasma membrane and the amplitude of mIPSC. Taken together, our findings identify a new anti-seizure target that provides a theoretical basis for the development of novel strategies for the prevention and treatment of epilepsy.

Abstract 1891: Identifying new therapeutic antibodies using the RenMice™ HiTS Platform

Abstract Over the past decade, an increasing number of therapeutic antibodies have been approved to target cancer. However, the process of identifying new therapeutic antibodies is routinely hampered by limitations in the traditional discovery process, including immune tolerance of highly homologous genes, and challenges with antibody sequence humanization, clone selection and drug efficacy and safety evaluation. RenMice™ HiTS (Hyperimmune Target Specific) is a collection of chromosome engineered mice with fully human immunoglobulin variable domains replacing mouse counterparts, each with a specific drug target gene knocked out. The goal of the RenMice™ HiTS platform is to overcome the challenges of traditional antibody drug discovery and increase the diversity of antibody paratopes and sequences which recognize functional epitopes. Immunization of target-specific RenMice™ generates a greater diversity of antibodies, including those that recognize the conserved regions between the antigen and the endogenous proteins of the immunized host. The platform is ideal for challenging targets, such as proteins with high homology between human and mouse, or multi-pass transmembrane proteins (eg. GPCRs/ion channels). The platform can be used to generate antibodies that cross-react with human, monkey, dog, and mouse targets, using a hybrid immunization strategy with both human and mouse/dog antigen. Generation of these species cross-reactive antibodies allows for high-throughput in vivo efficacy screening using wild-type mice. It is also possible to predict the preliminary response and toxicity in dogs with these cross-reactive antibodies. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of antibodies with better developability properties that recognize challenging targets and novel epitopes. Citation Format: Xiaoqian Zhang, Shujin Zhang, Tianquan Jin, Xin Ji, Jing Zhang. Identifying new therapeutic antibodies using the RenMice™ HiTS Platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1891.

SARS-CoV-2 ORF7a potently inhibits the antiviral effect of the host factor SERINC5

Serine Incorporator 5 (SERINC5), a cellular multipass transmembrane protein that is involved in sphingolipid and phosphatydilserine biogenesis, potently restricts a number of retroviruses, including Human Immunodeficiency Virus (HIV). SERINC5 is incorporated in the budding virions leading to the inhibition of virus infectivity. In turn, retroviruses, including HIV, encode factors that counteract the antiviral effect of SERINC5. While SERINC5 has been well studied in retroviruses, little is known about its role in other viral families. Due to the paucity of information regarding host factors targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), we evaluated the effect of SERINC proteins on SARS-CoV-2 infection. Here, we show SERINC5 inhibits SARS-CoV-2 entry by blocking virus-cell fusion, and SARS-CoV-2 ORF7a counteracts the antiviral effect of SERINC5 by blocking the incorporation of over expressed SERINC5 in budding virions.

RenMice™ HiTS platform enables identification of novel therapeutic antibodies

Abstract Despite an increase in approved cancer-targeting antibody drugs over the last decade, the process of identifying novel therapeutic antibodies is routinely hampered by limitations in the discovery process. Such barriers include immune tolerance of highly homologous genes, antibody sequence humanization, clone selection and models for drug efficacy/safety evaluation. To overcome these challenges and increase the diversity of antibody paratopes and sequences that recognize functional epitopes, we developed the RenMice™ HiTS (Hyperimmune Target Specific) Platform, which consists of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific target gene knocked out. Immunization of target-specific RenMice™ generates a sizeable diversity of antibodies, including those that recognize conserved regions between the antigen and the endogenous proteins of the immunized species. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins, such as GPCRs/ion channels, and can be used to generate antibodies that cross-react with human, monkey, dog, and mouse targets using a hybrid immunization strategy with both human and mouse/dog antigen. Generation of these species cross-reactive antibodies can be used for high-throughput in vivo efficacy screening in wild-type mice, and the preliminary response and toxicity can be assessed in dogs. Altogether, the RenMice™ HiTS platform facilitates the generation of antibodies that recognize novel epitopes and challenging targets while simultaneously allowing for a streamlined and successful preclinical phase based on in vivo efficacy and safety.

The Role of SDF‐1 in Signaling Differences Between CXCR4 and Atypical ACKR3 Chemokine Receptors

SDF‐1 is a ligand that plays a critical role in cancer metastases and is secreted in specialized somatic cells in the body. Its chemoattractant properties attract and induce migration of tumor cells located in other tissues in the body to the site in which SDF‐1 is secreted. This attraction and migration of tumor cells allows a new tumor to form at secondary locations within the body. Although the understanding of the function of SDF‐1 being secreted in certain regions of the body is not clear, we seek to understand the binding patterns of this ligand with its chemokine receptors CXCR4 and ACKR3 (also called CXCR7). Chemokine receptors are a subclass of G‐protein coupled receptors, which are multi‐pass transmembrane proteins with 7 TM domains. GPCR activation initiates signaling cascades within the targeted cell. Understanding which signaling pathway is favored in CXCR4 and ACKR3 can help us understand the function of SDF‐1 and the mechanism of secondary tumor formation and development. ACKR3 is considered an atypical receptor as it has been experimentally shown to not couple to Gi/o protein in many cells whereas CXCR4 can couple to either Gi/oprotein or β‐arrestin. ACKR3 strongly binds to SDF‐1 and can control the local levels of SDF‐1 available for binding to CXCR4. Both receptors can couple to β‐arrestins, which are proteins responsible for receptor internalization and potential G‐protein‐independent signaling. To understand the interplay between these two receptors in the context of their interactions with SDF‐1, Gi protein, and β‐arrestin, we have successfully generated structural models of these two receptors in apo form, as heterodimers (bound to only SDF‐1, only Gi protein, or only β‐arrestin), and as heterotrimers (bound to both SDF‐1 and Gi protein, or both SDF‐1 and β‐arrestin). These structural models have been relaxed in their physiological environment using a series of molecular dynamics (MD) simulations. Binding free energy analysis of the heterodimers is beginning to shed light on the signaling differences between the two receptors, what identifies ACKR3 as an atypical receptor, and how it sequesters excess SDF‐1 to modulate CXCR4 signaling.