Multi-spanning Transmembrane Proteins Research Articles

Structural bioinformatics studies of six human ABC transporters and their AlphaFold2-predicted water-soluble QTY variants.

Human ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins and perform diverse functions. Many of them are associated with multidrug resistance that often results in cancer treatment with poor outcomes. Here, we present the structural bioinformatics study of six human ABC membrane transporters with experimentally determined cryo-electron microscopy (CryoEM) structures including ABCB7, ABCC8, ABCD1, ABCD4, ABCG1, ABCG5, and their AlphaFold2-predicted water-soluble QTY variants. In the native structures, there are hydrophobic amino acids such as leucine (L), isoleucine (I), valine (V), and phenylalanine (F) in the transmembrane alpha helices. These hydrophobic amino acids are systematically replaced by hydrophilic amino acids glutamine (Q), threonine (T), and tyrosine (Y). Therefore, these QTY variants become water soluble. We also present the superposed structures of native ABC transporters and their water-soluble QTY variants. The superposed structures show remarkable similarity with root mean square deviations between 1.064 and 3.413Å despite significant (41.90-54.33%) changes to the protein sequence of the transmembrane domains. We also show the differences in hydrophobicity patches between the native ABC transporters and their QTY variants. We explain the rationale behind why the QTY membrane protein variants become water soluble. Our structural bioinformatics studies provide insight into the differences between the hydrophobic helices and hydrophilic helices and will likely further stimulate designs of water-soluble multispan transmembrane proteins and other aggregated proteins. The water-soluble ABC transporters may be useful as soluble antigens to generate therapeutic monoclonal antibodies for combating multidrug resistance in clinics.

A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli–based in vitro coupled transcription–translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.

The yeast dynamin-like GTPase Vps1 mediates Atg9 transport to the phagophore assembly site in Saccharomyces cerevisiae.

Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies.

Abstract 6036: Functional antibodies against multi span transmembrane proteins - revisiting the Warburg effect in cancer cells

Abstract Background: Alterations in tumor cell metabolism are one of the central processes guiding cancer progression. The increased glucose dependence of cancer cells, also known as the Warburg effect, opens the possibility to therapeutically target the glycolytic pathway and challenge the metabolic needs of tumor cells. We generated 24 monoclonal antibodies targeting the main glucose transporter on cancer cells - SLC2A1 (GLUT1). We evaluated the ability of the antibodies to affect cancer cell fitness alone and in combination with OXPHOS inhibitors to find clinically applicable therapeutics targeting tumor metabolism. virus like particle (VLP)-based immunization strategy (Kivi, et al., BMC Biotechnology, 2016). Antibodies were functionally characterized by 2-deoxyglucose uptake interference assay and sensitivity to antibody single and combination treatments were evaluated in various tumor cell lines. Metabolomics and oxygen consumption rates were examined as mechanistic endpoints for tumor cell lines and primary tumor samples. Results: The anti-SLC2A1 antibodies specifically bind to SLC2A1 with low nanomolar EC50 values and not to other glucose transporters. Antibody clones with functional properties inhibit glucose uptake leading to reduced metabolic activity and growth inhibition in a subset of cancer cell lines. A drastic cell proliferation inhibition is moreover observed in combination of anti-SLC2A1 antibodies and OXPHOS inhibitors metformin, phenformin or IACS-010759 in 2D cultures of colon, breast and pancreatic cancer cell lines. The inhibition of metabolic activity was further confirmed in an ex vivo primary patient tissue samples highlighting the putative translational applicability of our antibodies. Conclusions: This is the first study to provide highly specific antibodies bocking the function of SLC2A1 transporter and demonstrate the proof of principle for inhibiting complex muti-pass membrane transporters with antibody therapeutics. While the developed anti-SLC2A1 antibodies could be efficacious in some indications as single agents, appreciable clinical activity could be obtained by the combined use with OXPHOS inhibitors. Citation Format: Siret Tahk, Kai Virumäe, Paule Hermet, Korneelia Anton, Maiken Abel, Denis Belitškin, Luciano Galdieri, Steven Garner, Jillian Krings, Kaleb Collver, Emily Hoehn, Brendan Lahm, Kaitlyne Powers, Tuuli Käämbre, Alastair J. King, Francisca Neethling, Anu Planken, Mart Ustav, Andres Männik, Mart Ustav. Functional antibodies against multi span transmembrane proteins - revisiting the Warburg effect in cancer cells [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 6036.

SERINC5 restricts influenza virus infectivity.

SERINC5 is a multi-span transmembrane protein that is incorporated into HIV-1 particles in producing cells and inhibits HIV-1 entry. Multiple retroviruses like HIV-1, equine infectious anemia virus and murine leukemia virus are subject to SERINC5 inhibition, while HIV-1 pseudotyped with envelope glycoproteins of vesicular stomatitis virus and Ebola virus are resistant to SERINC5. The antiviral spectrum and the underlying mechanisms of SERINC5 restriction are not completely understood. Here we show that SERINC5 inhibits influenza A virus infection by targeting virus-cell membrane fusion at an early step of infection. Further results show that different influenza hemagglutinin (HA) subtypes exhibit diverse sensitivities to SERINC5 restriction. Analysis of the amino acid sequences of influenza HA1 strains indicates that HA glycosylation sites correlate with the sensitivity of influenza HA to SERINC5, and the inhibitory effect of SERINC5 was lost when certain HA glycosylation sites were mutated. Our study not only expands the antiviral spectrum of SERINC5, but also reveals the role of viral envelope glycosylation in resisting SERINC5 restriction.

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.

Comparing 2 crystal structures and 12 AlphaFold2-predicted human membrane glucose transporters and their water-soluble glutamine, threonine and tyrosine variants.

Membrane transporters including glucose transporters (GLUTs) are involved in cellular energy supplies, cell metabolism and other vital biological activities. They have also been implicated in cancer proliferation and metastasis, thus they represent an important target in combatting cancer. However, membrane transporters are very difficult to study due to their multispan transmembrane properties. The new computational tool, AlphaFold2, offers highly accurate predictions of three-dimensional protein structures. The glutamine, threonine and tyrosine (QTY) code provides a systematic method of rendering hydrophobic sequences into hydrophilic ones. Here, we present computational studies of native integral membrane GLUTs with 12 transmembrane helical segments determined by X-ray crystallography and CryoEM, comparing the AlphaFold2-predicted native structure to their water-soluble QTY variants predicted by AlphaFold2. In the native structures of the transmembrane helices, there are hydrophobic amino acids leucine (L), isoleucine (I), valine (V) and phenylalanine (F). Applying the QTY code, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, glutamine (Q), threonine (T) and tyrosine (Y) rendering them water-soluble. We present the superposed structures of native GLUTs and their water-soluble QTY variants. The superposed structures show remarkable similar residue mean square distance values between 0.47 and 3.6Å (most about 1-2Å) despite >44% transmembrane amino acid differences. We also show the differences of hydrophobicity patches between the native membrane transporters and their QTY variants. We explain the rationale why the membrane protein QTY variants become water-soluble. Our study provides insight into the differences between the hydrophobic helices and hydrophilic helices, and offers confirmation of the QTY method for studying multispan transmembrane proteins and other aggregated proteins through their water-soluble variants.

Probing the Membrane Environment of Plasma Membrane Proteins: A Micropatterning Approach

It is well established that lipids and proteins are not just independent components of the plasma membrane of eukaryotic cells but that their arrangement, dynamics and function are interdependent. Besides specific lipid-protein interaction, transmembrane proteins are thought to bind a shell of annular lipids, which are more or less tightly associated with the proteins. Furthermore, highly ordered nanoscopic membrane domains have been proposed to act to compartmentalize proteins and their interactions, but have thus far not been directly observed. Here, we use a combination of single molecule tracking and protein micropatterning to examine these interactions directly in the plasma membrane of living cells. In our experimental approach, different proteins of interest are immobilized within defined patterns in the plasma membrane, where they act as steric obstacles to the diffusion of lipid tracers and thus locally decrease their mobility. In the presence of lipid-protein interactions of any type, lipid mobility will be even further decreased within protein patterns. We used different types of proteins for patterning - single- and multi-spanning transmembrane proteins, a multi-subunit protein and a GPI-anchored protein. For all proteins we found that lipid tracer diffusion within protein patterns was slowed down only by steric hindrance since the size of the protein “sensed” by the diffusing lipid corresponded well with the size estimated from the protein crystal structure. These findings indicate that none of the examined proteins influence their membrane environment beyond their physical size, neither via tightly associated annular lipids nor via more ordered membrane domains.

Insights into the Roles of the Sideroflexins/SLC56 Family in Iron Homeostasis and Iron-Sulfur Biogenesis.

Sideroflexins (SLC56 family) are highly conserved multi-spanning transmembrane proteins inserted in the inner mitochondrial membrane in eukaryotes. Few data are available on their molecular function, but since their first description, they were thought to be metabolite transporters probably required for iron utilization inside the mitochondrion. Such as numerous mitochondrial transporters, sideroflexins remain poorly characterized. The prototypic member SFXN1 has been recently identified as the previously unknown mitochondrial transporter of serine. Nevertheless, pending questions on the molecular function of sideroflexins remain unsolved, especially their link with iron metabolism. Here, we review the current knowledge on sideroflexins, their presumed mitochondrial functions and the sparse—but growing—evidence linking sideroflexins to iron homeostasis and iron-sulfur cluster biogenesis. Since an imbalance in iron homeostasis can be detrimental at the cellular and organismal levels, we also investigate the relationship between sideroflexins, iron and physiological disorders. Investigating Sideroflexins’ functions constitutes an emerging research field of great interest and will certainly lead to the main discoveries of mitochondrial physio-pathology.

ANXA6 suppresses the tumorigenesis of cervical cancer through autophagy induction.

BackgroundAutophagy is an intracellular degradation pathway conserved in eukaryotes. ANXA6 (annexin A6) belongs to a family of calcium‐dependent membrane and phospholipid‐binding proteins. Here, we identify ANXA6 as a newly synthesized protein in starvation‐induced autophagy and validate it as a novel autophagy modulator that regulates autophagosome formation.Results ANXA6 knockdown attenuates starvation‐induced autophagy, while restoration of its expression enhances autophagy. GO (gene ontology) analysis of ANXA6 targets showed that ANXA6 interacts with many RAB GTPases and targets endocytosis and phagocytosis pathways, indicating that ANXA6 exerts its function through protein trafficking. ATG9A (autophagy‐related 9A) is the sole multispanning transmembrane protein and its trafficking through recycling endosomes is an essential step for autophagosome formation. Our results showed that ANXA6 enables appropriate ATG9A+ vesicle trafficking from endosomes to autophagosomes through RAB proteins or F‐actin. In addition, restoration of ANXA6 expression suppresses mTOR (mammalian target of rapamycin) activity through the inhibition of the PI3K (phosphoinositide 3‐kinase)‐AKT and ERK (extracellular signal‐regulated kinase) signaling pathways, which is a negative regulator of autophagy. Functionally, ANXA6 expression is correlated with LC3 (microtubule‐associated protein 1 light chain 3) expression in cervical cancer, and ANXA6 inhibits tumorigenesis through autophagy induction.ConclusionsOur results reveal an important mechanism for ANXA6 in tumor suppression and autophagy regulation.

TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis.

Wnt signaling through the Frizzled (FZD) family of serpentine receptors is essential for embryogenesis and homeostasis, and stringent control of the FZD protein level is critical for stem cell regulation. Through CRISPR/Cas9 genome-wide screening in human cells, we identified TMEM79/MATTRIN, an orphan multi-span transmembrane protein, as a specific inhibitor of Wnt/FZD signaling. TMEM79 interacts with FZD during biogenesis and promotes FZD degradation independent of ZNRF3/RNF43 ubiquitin ligases (R-spondin receptors). TMEM79 interacts with ubiquitin-specific protease 8 (USP8), whose activating mutations underlie human tumorigenesis. TMEM79 specifically inhibits USP8 deubiquitination of FZD, thereby governing USP8 substrate specificity and promoting FZD degradation. Tmem79 and Usp8 genes have a pre-bilaterian origin, and Tmem79 inhibition of Usp8 and Wnt signaling is required for anterior neural development and gastrulation in Xenopus embryos. TMEM79 is a predisposition gene for Atopic dermatitis, suggesting deregulation of Wnt/FZD signaling a possible cause for this most common yet enigmatic inflammatory skin disease.

A Chloroplast COR413 Protein From Physcomitrella patens Is Required for Growth Regulation Under High Light and ABA Responses.

COR413 genes belong to a poorly characterized group of plant-specific cold-regulated genes initially identified as part of the transcriptional activation machinery of plants during cold acclimation. They encode multispanning transmembrane proteins predicted to target the plasma membrane or the chloroplast inner membrane. Despite being ubiquitous throughout the plant kingdom, little is known about their biological function. In this study, we used reverse genetics to investigate the relevance of a predicted chloroplast localized COR413 protein (PpCOR413im) from the moss Physcomitrella patens in developmental and abiotic stress responses. Expression of PpCOR413im was strongly induced by abscisic acid (ABA) and by various environmental stimuli, including low temperature, hyperosmosis, salinity and high light. In vivo subcellular localization of PpCOR413im-GFP fusion protein revealed that this protein is localized in chloroplasts, confirming the in silico predictions. Loss-of-function mutants of PpCOR413im exhibited growth and developmental alterations such as growth retardation, reduced caulonema formation and hypersensitivity to ABA. Mutants also displayed altered photochemistry under various abiotic stresses, including dehydration and low temperature, and exhibited a dramatic growth inhibition upon exposure to high light. Disruption of PpCOR413im also caused altered chloroplast ultrastructure, increased ROS accumulation, and enhanced starch and sucrose levels under high light or after ABA treatment. In addition, loss of PpCOR413im affected both nuclear and chloroplast gene expression in response to ABA and high light, suggesting a role for this gene downstream of ABA in the regulation of growth and environmental stress responses. Developmental alterations exhibited by PpCOR413im knockout mutants had remarkable similarities to those exhibited by hxk1, a mutant lacking a major chloroplastic hexokinase, an enzyme involved in energy homeostasis. Based on these findings, we propose that PpCOR413im is involved in coordinating energy metabolism with ABA-mediated growth and developmental responses.

Peripheral myelin protein 22 preferentially partitions into ordered phase membrane domains

The ordered environment of cholesterol-rich membrane nanodomains is thought to exclude many transmembrane (TM) proteins. Nevertheless, some multispan helical transmembrane proteins have been proposed to partition into these environments. Here, giant plasma membrane vesicles (GPMVs) were employed to quantitatively show that the helical tetraspan peripheral myelin protein 22 (PMP22) exhibits a pronounced preference for, promotes the formation of, and stabilizes ordered membrane domains. Neither S-palmitoylation of PMP22 nor its putative cholesterol binding motifs are required for this preference. In contrast, Charcot-Marie-Tooth disease-causing mutations that disrupt the stability of PMP22 tertiary structure reduce or eliminate this preference in favor of the disordered phase. These studies demonstrate that the ordered phase preference of PMP22 derives from global structural features associated with the folded form of this protein, providing a glimpse at the structural factors that promote raft partitioning for multispan helical membrane proteins.

Individualized management of genetic diversity in Niemann-Pick C1 through modulation of the Hsp70 chaperone system.

Genetic diversity provides a rich repository for understanding the role of proteostasis in the management of the protein fold in human biology. Failure in proteostasis can trigger multiple disease states, affecting both human health and lifespan. Niemann-Pick C1 (NPC1) disease is a rare genetic disorder triggered by mutations in NPC1, a multi-spanning transmembrane protein that is trafficked through the exocytic pathway to late endosomes (LE) and lysosomes (Ly) (LE/Ly) to globally manage cholesterol homeostasis. Defects triggered by >300 NPC1 variants found in the human population inhibit export of NPC1 protein from the endoplasmic reticulum (ER) and/or function in downstream LE/Ly, leading to cholesterol accumulation and onset of neurodegeneration in childhood. We now show that the allosteric inhibitor JG98, that targets the cytosolic Hsp70 chaperone/co-chaperone complex, can significantly improve the trafficking and post-ER protein level of diverse NPC1 variants. Using a new approach to model genetic diversity in human disease, referred to as variation spatial profiling, we show quantitatively how JG98 alters the Hsp70 chaperone/co-chaperone system to adjust the spatial covariance (SCV) tolerance and set-points on an amino acid residue-by-residue basis in NPC1 to differentially regulate variant trafficking, stability, and cholesterol homeostasis, results consistent with the role of BCL2-associated athanogene family co-chaperones in managing the folding status of NPC1 variants. We propose that targeting the cytosolic Hsp70 system by allosteric regulation of its chaperone/co-chaperone based client relationships can be used to adjust the SCV tolerance of proteostasis buffering capacity to provide an approach to mitigate systemic and neurological disease in the NPC1 population.

Characteristics and Functions of the Yip1 Domain Family (YIPF), Multi-Span Transmembrane Proteins Mainly Localized to the Golgi Apparatus.

Yip1 domain family (YIPF) proteins are multi-span, transmembrane proteins mainly localized in the Golgi apparatus. YIPF proteins have been found in virtually all eukaryotes, suggesting that they have essential function(s). Saccharomyces cerevisiae contains four YIPFs: Yip1p, Yif1p, Yip4p, and Yip5p. Early analyses in S. cerevisiae indicated that Yip1p and Yif1p bind to each other and play a role in budding of transport vesicles and/or fusion of vesicles to target membranes. However, the molecular basis of their functions remains unclear. Analysis of YIPF proteins in mammalian cells has yielded significant clues about the function of these proteins. Human cells have nine family members that appear to have overlapping functions. These YIPF proteins are divided into two sub-families: YIPFα/Yip1p and YIPFβ/Yif1p. A YIPFα molecule forms a complex with a specific partner YIPFβ molecule. In the most broadly hypothesized scenario, a basic tetramer complex is formed from two molecules of each partner YIPF protein, and this tetramer forms a higher order oligomer. Three distinct YIPF protein complexes are formed from pairs of YIPFα and YIPFβ proteins. These are differently localized in either the early, middle, or late compartments of the Golgi apparatus and are recycled between adjacent compartments. Because a YIPF protein is predicted to have five transmembrane segments, a YIPF tetramer complex is predicted to have 20 transmembrane segments. This high number of transmembrane segments suggests that YIPF complexes function as channels, transporters, or transmembrane receptors. Here, the evidence from functional studies of YIPF proteins obtained during the last two decades is summarized and discussed.

MBOAT7 is anchored to endomembranes by six transmembrane domains

Membrane bound O-acyltransferase domain- containing 7 (MBOAT7, also known as LPIAT1) is a protein involved in the acyl chain remodeling of phospholipids via the Lands’ cycle. The MBOAT7 is a susceptibility risk genetic locus for non-alcoholic fatty liver disease (NAFLD) and mental retardation. Although it has been shown that MBOAT7 is associated to membranes, the MBOAT7 topology remains unknown. To solve the topological organization of MBOAT7, we performed: A) solubilization of the total membrane fraction of cells overexpressing the recombinant MBOAT7-V5, which revealed MBOAT7 is an integral protein strongly attached to endomembranes; B) in silico analysis by using 22 computational methods, which predicted the number and localization of transmembrane domains of MBOAT7 with a range between 5 and 12; C) in vitro analysis of living cells transfected with GFP-tagged MBOAT7 full length and truncated forms, using a combination of Western Blotting, co-immunofluorescence and Fluorescence Protease Protection (FPP) assay; D) in vitro analysis of living cells transfected with FLAG-tagged MBOAT7 full length forms, using a combination of Western Blotting, selective membrane permeabilization followed by indirect immunofluorescence. All together, these data revealed that MBOAT7 is a multispanning transmembrane protein with six transmembrane domains. Based on our model, the predicted catalytic dyad of the protein, composed of the conserved asparagine in position 321 (Asn-321) and the preserved histidine in position 356 (His-356), has a lumenal localization. These data are compatible with the role of MBOAT7 in remodeling the acyl chain composition of endomembranes.

Proteomic Analysis of the Function of a Novel Cold-Regulated Multispanning Transmembrane Protein COR413-PM1 in Arabidopsis.

The plasma membrane is the first subcellular organ that senses low temperature, and it includes some spanning transmembrane proteins that play important roles in cold regulation. COR413-PM1 is a novel multispanning transmembrane cold-regulated protein; however, the related functions are not clear in Arabidopsis. We found the tolerance to freezing stress of cor413-pm1 was lower than wild-type (WT). A proteomics method was used to analyze the differentially abundant proteins (DAPs) between cor413-pm1 and WT. A total of 4143 protein groups were identified and 3139 were accurately quantitated. The DAPs associated with COR413-PM1 and freezing treatment were mainly involved in the metabolism of fatty acids, sugars, and purine. Quantitative real-time PCR (qRT-PCR) confirmed the proteomic analysis results of four proteins: fatty acid biosynthesis 1 (FAB1) is involved in fatty acid metabolism and might affect the plasma membrane structure; fructokinase 3 (FRK3) and sucrose phosphate synthase A1 (SPSA1) play roles in sugar metabolism and may influence the ability of osmotic adjustment under freezing stress; and GLN phosphoribosyl pyrophosphate amidotransferase 2 (ASE2) affects freezing tolerance through purine metabolism pathways. In short, our results demonstrate that the multispanning transmembrane protein COR413-PM1 regulates plant tolerance to freezing stress by affecting the metabolism of fatty acids, sugars, and purine in Arabidopsis.

CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47-/- hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47-/- mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47-/- mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47-/- mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47-/- mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47-/- mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47-/- mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

Localization to detergent-resistant membranes and HIV-1 core entry inhibition correlate with HIV-1 restriction by SERINC5

SERINC5(S5) is a multi-span transmembrane protein that potently blocks the infectivity of HIV-1 produced by human T-cells. The ability of S5 to restrict infectivity correlates with its presence in the virion, but the exact mechanism by which S5 restricts HIV-1 is unknown. Here we tested whether the core from HIV-1 virions containing S5 is delivered to the cytoplasm. Using the “fate of the capsid” assay, we demonstrated that the viral core of S5-restricted HIV-1 does not reach the cytoplasm of target cells, suggesting a block in the delivery of the core to the cytoplasm. In agreement with evidence suggesting that the viral determinants for S5 restriction map to the envelope of HIV-1, we observed that S5 induces conformational changes to the HIV-1 envelope. Further, we demonstrated that S5 localizes to detergent-resistant membranes (DRMs), as has been shown previously for the HIV-1 envelope in producer cells. In order to identify the determinants of S5 restriction, we explored the ability of all human SERINC proteins to restrict HIV-1. In contrast to human S5, we observed that human SERINC2(S2) did not restrict HIV-1, and was inefficiently incorporated into HIV-1 virions when compared to S5. Experiments using S5-S2 chimeric proteins revealed two functional domains for restriction: one necessary for S5 incorporation into virions, which does not seem to be necessary for restriction, and a second one necessary to change the HIV-1 envelope conformation, localize to DRMs, and block infection.

Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.