Human Transmembrane Protein Research Articles

Discovery of Carbonic Anhydrase 9 as a Novel CLEC2 Ligand in a Cellular Interactome Screen

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin’s lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.

In silico analysis of aptamer-RNA conjugate interactions with human transferrin receptor

The human transmembrane protein Transferrin Receptor-1 is regarded as a promising target for the systemic delivery of therapeutic agents, particularly of nucleic acid therapeutics, such as short double stranded RNAs. This ubiquitous receptor is involved in cellular iron uptake, keeping intracellular homeostasis. It is overexpressed in multiple cancer cell types and is internalized via clathrin-mediated endocytosis. In previous studies, a human transferrin receptor-1 RNA aptamer, identified as TR14 ST1–3, was shown to be capable of effectively internalizing into cells in culture and to deliver small, double stranded RNAs in vitro and in vivo, via systemic administration.To understand, at the molecular level, the aptamer binding to the receptor and the impact of conjugation with the therapeutic RNA, a multi-level in silico protocol was employed, including protein-aptamer docking, molecular dynamics simulations and free energy calculations. The competition for the binding pocket, between the aptamer and the natural ligand human Transferrin, was also evaluated.The results show that the aptamer binds to the same region as Transferrin, with residues from the helical domain showing a critical role. Moreover, the conjugation to the therapeutic RNA, was shown not to affect aptamer binding. Overall, this study provides an atomic-level understanding of aptamer association to human Transferrin Receptor-1 and of its conjugation with a short model-therapeutic RNA, providing also important clues for futures studies aiming to deliver other oligonucleotide-based therapeutics via Transferrin Receptor.

Structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1) reveals the conformational flexibility of its lipid binding domain.

In Caenorhabditis elegans, inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNAi defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the ChUP family. Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of sid gene products-hSIDT1 and hSIDT2-mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain exists as two modules-a flexible lipid binding domain and a rigid transmembrane domain core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in ChUP family members.

Structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1) reveals the conformational flexibility of its lipid binding domain.

In C. elegans, inter-cellular transport of the small non-coding RNA causing systemic RNA interference (RNAi) is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNA interference-defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the cholesterol uptake family (ChUP). Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of sid gene products - hSIDT1 and hSIDT2 - mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain (ECD) of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain (TMD) exists as two modules - a flexible lipid binding domain (LBD) and a rigid TMD core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in cholesterol uptake (ChUP) family members.

Sorting of secretory proteins at the trans-Golgi network by human TGN46

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS – a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.

Sorting of secretory proteins at the trans-Golgi network by human TGN46.

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.

IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

IFITM1 and IFITM3 Proteins Inhibit the Infectivity of Progeny HIV-1 without Disrupting Envelope Glycoprotein Clusters.

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane.

UniTmp: unified resources for transmembrane proteins.

The UNIfied database of TransMembrane Proteins (UniTmp) is a comprehensive and freely accessible resource of transmembrane protein structural information at different levels, from localization of protein segments, through the topology of the protein to the membrane-embedded 3D structure. We not only annotated tens of thousands of new structures and experiments, but we also developed a new system that can serve these resources in parallel. UniTmp is a unified platform that merges TOPDB (Topology Data Bank of Transmembrane Proteins), TOPDOM (database of conservatively located domains and motifs in proteins), PDBTM (Protein Data Bank of Transmembrane Proteins)and HTP (Human Transmembrane Proteome) databases and provides interoperability between the incorporated resources and an easy way to keep them regularly updated. The current update contains 9235 membrane-embedded structures, 9088 sequences with 536035 topology-annotated segmentsand 8692 conservatively localized protein domains or motifs as well as 5466 annotated human transmembrane proteins. The UniTmp database can be accessed at https://www.unitmp.org.

Golimumab for Polyarticular Juvenile Idiopathic Arthritis and Psoriatic Arthritis: Pharmacologic and Clinical Considerations.

Psoriatic arthritis is a chronic debilitating autoimmune condition, and when diagnosed in patients before the age of eighteen, it is considered pediatric polyarticular juvenile idiopathic arthritis. Monoarticular or polyarticular psoriatic arthritis can be distinguished from other arthropathies by its unique cutaneous manifestations. With numerous treatments already in clinical practice, there are numerous options for treatment. The current literature indicates an elevated level of tumor necrosis factor is present in the epidermis of patients with psoriatic arthritis when compared with the general population. For this reason, anti-tumor necrosis factor therapies have become a hallmark option for psoriatic arthritis patients. Golimumab, a human monoclonal antibody tumor necrosis factor-alpha (TNF-a) receptor antagonist, was chosen as the focus therapy for this investigation. The mechanism of action behind anti-tumor necrosis factor-alpha blockers involves the binding of human TNF-a soluble and transmembrane proteins to competitively inhibit TNF-a from binding to its cellular receptors. The present investigation evaluated current treatment options available for both juvenile- and adult-onset psoriatic arthritis and compared them with the efficacy seen with golimumab use. Pediatric patients included children ages 2-17, while adult populations included adults 18-83 years old. The Food and Drug Administration has approved golimumab for the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, and polyarticular juvenile idiopathic arthritis. The results of four different studies reporting on the therapeutic effects and adverse events of golimumab use in psoriatic arthritis, juvenile psoriatic arthritis, juvenile idiopathic arthritis, and juvenile polyarticular arthritis were used for comparison. The meta-analysis referenced studies including children ages 2-17 with no reference mentioning children less than age 2. Based on the results of each study, it can be concluded that golimumab, a human monoclonal antibody that prevents the activation of cellular inflammatory reactions when it binds to the TNF-a receptor, is an effective option for patients with active psoriatic arthritis and psoriatic juvenile idiopathic arthritis and for patients who are no longer responding to their current treatment with adalimumab. Each study also reported minimal adverse events associated with golimumab use, and the drug can be safely used in the pediatric population.

Employing nano-SnFe2O4 in the synthesis of arylbenzimidazoles, the computational study of the reaction mechanism, and examining the products as putative human transmembrane protein activators

Employing nano-SnFe2O4 in the synthesis of arylbenzimidazoles, the computational study of the reaction mechanism, and examining the products as putative human transmembrane protein activators

IFITM2 Presents Antiviral Response through Enhancing Type I IFN Signaling Pathway.

Interferon (IFN) helps cells fight viral infections by further inducing the expression of many downstream IFN-stimulated genes (ISGs). Human interferon-inducible transmembrane proteins (IFITM) are one of these ISGs. The antiviral function of human IFITM1, IFITM2, and IFITM3 are well known. In this study, we report that IFITM can significantly inhibit EMCV infectivity in HEK293 cells. Overexpression of IFITM proteins could promote IFN-β production. Meanwhile, IFITMs facilitated type I IFN signaling pathway adaptor MDA5 expression. We detected the binding of IFITM2 to MDA5 in a co-immunoprecipitation assay. It was also found that the ability of IFITM2 to activate IFN-β was significantly inhibited after interfering with MDA5 expression, suggesting that MDA5 may play an important role in the activation of the IFN-β signaling pathway by IFITM2. Moreover, the N-terminal domain plays an active role in the antiviral activity and the activation of IFN-β by IFITM2. These findings suggest that IFITM2 plays a vital role in antiviral signaling transduction. In addition, a positive feed-forward loop between IFITM2 and type I IFN establishes a key role for IFITM2 in enforcing innate immune responses.

Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody

The early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies.

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Next-generation sequencing of human genomes reveals millions of missense variants, some of which may lead to loss of protein function and ultimately disease. Here, we investigate missense variants in membrane proteins—key drivers in cell signaling and recognition. We find enrichment of pathogenic variants in the transmembrane region across 19,000 functionally classified variants in human membrane proteins. To accurately predict variant consequences, one fundamentally needs to understand the underlying molecular processes. A key mechanism underlying pathogenicity in missense variants of soluble proteins has been shown to be loss of stability. Membrane proteins, however, are widely understudied. Here, we interpret variant effects on a larger scale by performing structure-based estimations of changes in thermodynamic stability using a membrane-specific energy function and analyses of sequence conservation during evolution of 15 transmembrane proteins. We find evidence for loss of stability being the cause of pathogenicity in more than half of the pathogenic variants, indicating that this is a driving factor also in membrane-protein-associated diseases. Our findings show how computational tools aid in gaining mechanistic insights into variant consequences for membrane proteins. To enable broader analyses of disease-related and population variants, we include variant mappings for the entire human proteome.

Use of Tofacitinib for infant-onset STING-associated vasculopathy: A case report from China

Stimulator of interferon gene (STING)-associated vasculopathy with onset in infancy (SAVI), caused by gain-of-function mutations in human transmembrane protein 173 (TMEM173), is characterized by widespread chronic inflammation primarily affecting the skin and lungs. Although SAVI is an inflammatory disease, typical anti-inflammatory agents have limited or no effect. A 1-year-old boy presented with recurrent facial rashes since he was 8 months. Moreover, he suffered from recurrent oral ulcers, chronic cough, and failure to thrive. Laboratory parameters showed elevated erythrocyte sedimentation rate (ESR) and immunoglobulin levels. Chest high-resolution computed tomography (HRCT) showed interstitial lung disease (ILD). Whole-exome sequencing revealed a heterozygous mutation in the TMEM173 gene (c.463G > A, p.V155M). Ultimately, the patient was diagnosed with SAVI. Tofacitinib was initiated at the age of 19 months, resulting in the alleviation of facial rashes and improvement of ILD within 3 months. SAVI is a difficult-to-treat type I interferonopathy. We hope that JAKi treatment will prove valuable for SAVI patients.

Single-gene knockout-coupled omics analysis identifies C9orf85 and CXorf38 as two uncharacterized human proteins associated with ZIP8 malfunction.

Human transmembrane protein metal cation symporter ZIP8 (SLC39A8) is a member of the solute carrier gene family responsible for intracellular transportation of essential micronutrients, including manganese, selenium, and zinc. Previously, we established a ZIP8-knockout (KO) human cell model using the CRISPR/Cas9 system and explored how the expression of ZIP8 could possibly contribute to a wide range of human diseases. To further assess the biophysiological role of ZIP8, in the current study, we employed isobaric tags for relative and absolute quantitation (iTRAQ) and detected the changes of the proteome in ZIP8-KO cells (proteomic data are available via ProteomeXchange with identifier PXD036680). A total of 286 differentially expressed proteins (206 downregulated and 80 upregulated proteins) were detected in the ZIP8-KO cell model, and subsequent bioinformatics analyses (GO, KEGG, KOG, and PPI) were performed on these proteins. Interestingly, four "uncharacterized" proteins (proteins with unknown biological function) were identified in the differentially expressed proteins: C1orf198, C9orf85, C17orf75, and CXorf38-all of which were under-expressed in the ZIP8-KO cells. Notably, C9orf85 and CXorf38 were amongst the top-10 most downregulated proteins, and their expressions could be selectively induced by essential micronutrients. Furthermore, clinical-based bioinformatic analysis indicated that positive correlations between the gene expressions of ZIP8 and C9orf85 or CXorf38 were observed in multiple cancer types. Overall, this study reveals the proteomic landscape of cells with impaired ZIP8 and uncovers the potential relationships between essential micronutrients and uncharacterized proteins C9orf85 and CXorf38. The differentially expressed proteins identified in ZIP8-KO cells could be the potential targets for diagnosing and/or treating human ZIP8-associated diseases, including but not limited to malnutrition, viral infection, and cancers.

Retracted: Human Interferon Inducible Transmembrane Protein 3 (IFITM3) Inhibits Influenza Virus A Replication and Inflammation by Interacting with ABHD16A.

[This retracts the article DOI: 10.1155/2021/6652147.].

Unbinding of hACE2 and inhibitors from the receptor binding domain of SARS-CoV-2 spike protein

The first direful biomolecular event leading to COVID-19 disease is the SARS-CoV-2 virus surface spike (S) protein-mediated interaction with the human transmembrane protein, angiotensin-converting enzyme 2 (hACE2). Prevention of this interaction presents an attractive alternative to thwart SARS-CoV-2 replications. The development of monoclonal antibodies (mAbs) in the convalescent plasma treatment, nanobody, and designer peptides, which recognizes epitopes that overlap with hACE2 binding sites in the receptor-binding domain (RBD) of S protein (S/RBD) and thereby blocking the infection has been the center stage of therapeutic research. Here we report atomistic and reliable in silico structure-energetic features of the S/RBD interactions with hACE2 and its two inhibitors (convalescent mAb, B38, and an alpaca nanobody, Ty1). The discovered potential of mean forces exhibits free energy basin and barriers along the interaction pathways, providing sufficient molecular insights to design a B38 mutant and a Ty1-based peptide with higher binding capacity. While the mutated B38 forms a 60-fold deeper free energy minimum, the designer peptide (Ty1-based) constitutes 38 amino acids and is found to form a 100-fold deeper free energy minimum in the first binding basin than their wild-type variants in complex with S/RBD. Our strategy may help to design more efficacious biologics towards therapeutic intervention against the current raging pandemic. Communicated by Ramaswamy H. Sarma

Predicting the multi-label protein subcellular localization through multi-information fusion and MLSI dimensionality reduction based on MLFE classifier.

MotivationMulti-label (ML) protein subcellular localization (SCL) is an indispensable way to study protein function. It can locate a certain protein (such as the human transmembrane protein that promotes the invasion of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) or expression product at a specific location in a cell, which can provide a reference for clinical treatment of diseases such as coronavirus disease 2019 (COVID-19).ResultsThe article proposes a novel method named ML-locMLFE. First of all, six feature extraction methods are adopted to obtain protein effective information. These methods include pseudo amino acid composition, encoding based on grouped weight, gene ontology, multi-scale continuous and discontinuous, residue probing transformation and evolutionary distance transformation. In the next part, we utilize the ML information latent semantic index method to avoid the interference of redundant information. In the end, ML learning with feature-induced labeling information enrichment is adopted to predict the ML protein SCL. The Gram-positive bacteria dataset is chosen as a training set, while the Gram-negative bacteria dataset, virus dataset, newPlant dataset and SARS-CoV-2 dataset as the test sets. The overall actual accuracy of the first four datasets are 99.23%, 93.82%, 93.24% and 96.72% by the leave-one-out cross validation. It is worth mentioning that the overall actual accuracy prediction result of our predictor on the SARS-CoV-2 dataset is 72.73%. The results indicate that the ML-locMLFE method has obvious advantages in predicting the SCL of ML protein, which provides new ideas for further research on the SCL of ML protein.Availability and implementationThe source codes and datasets are publicly available at https://github.com/QUST-AIBBDRC/ML-locMLFE/.Supplementary information Supplementary data are available at Bioinformatics online.

Integrative overview of IFITMs family based on Bioinformatics analysis.

Human interferon-induced transmembrane proteins (IFITMs) family is a multi-functional biomacromolecule family playing a critical role in various physiological processes, such as, antiviral immunity, tumor suppression, and bone formation. Although there are many studies proving that a subset of tumors strongly links to the changes of IFITMs, the link between different IFITMs mutant types and diverse tumors has not been studied thoroughly. To investigate the law of expression among IFITMs internal members and the linking of IFITMs mutant types and cancers, online databases were used to pool together relevant data for bioinformatics analysis. Here, we summarize mutations, expression, and functions of human IFITMs, analyze diverse expression levels of IFITMs in physiological and pathological tissues, predict protein-protein interaction (PPI) networks, and target miRNAs and relevant signaling pathways of IFITMs. The results show that IFITM1, IFITM2, and IFITM3 have similar motif pattern constructions and physiological functions, while IFITM5 and IFITM10 show far diversity from them. Particularly, IFITM1-3, in conjunction with interacting proteins, is strongly related to development and overall survival rates of a portion of cancers, including renal cancer and uveal melanoma (UVM). This trait may make IFITM1-3 become a prognostic marker of cancers. Meanwhile, hsa_circ_0116375 has been found as the common circRNA for IFITM2, IFITM3, IFITM5, and IFITM10.