scholarly journals Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains

2016 ◽  
Vol 0 (0) ◽  
Author(s):  
Frederike Schmidt ◽  
Miryam Müller ◽  
Johannes Prox ◽  
Philipp Arnold ◽  
Caroline Schönherr ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Amyloid Β ◽  
Type I ◽  
Direct Binding ◽  
Small Intestinal ◽  
Split Ubiquitin ◽  
Biological Methods ◽  
Two Hybrid

AbstractMeprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer’s disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

scholarly journals Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains

2016 ◽  
Vol 397 (9) ◽  
pp. 857-869 ◽  
Author(s):  
Frederike Schmidt ◽  
Miryam Müller ◽  
Johannes Prox ◽  
Philipp Arnold ◽  
Caroline Schönherr ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Amyloid Β ◽  
Type I ◽  
Direct Binding ◽  
Small Intestinal ◽  
Split Ubiquitin ◽  
Biological Methods ◽  
Two Hybrid

Abstract Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer’s disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

scholarly journals TMEFF2: A Transmembrane Proteoglycan with Multifaceted Actions in Cancer and Disease

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3862
Author(s):  
Motasim Masood ◽  
Stefan Grimm ◽  
Mona El-Bahrawy ◽  
Ernesto Yagüe
Keyword(s):  
Prostate Cancer ◽  
Cancer Progression ◽  
Transmembrane Protein ◽  
Intracellular Localization ◽  
Amyloid Β ◽  
Inverse Correlation ◽  
Response To Therapy ◽  
Endocrine Function ◽  
Type I ◽  
Amyloid Β Protein

Transmembrane protein with an EGF-like and two Follistatin-like domains 2 (TMEFF2) is a 374-residue long type-I transmembrane proteoglycan which is proteolytically shed from the cell surface. The protein is involved in a range of functions including metabolism, neuroprotection, apoptosis, embryonic development, onco-suppression and endocrine function. TMEFF2 is methylated in numerous cancers, and an inverse correlation with the stage, response to therapy and survival outcome has been observed. Moreover, TMEFF2 methylation increases with breast, colon and gastric cancer progression. TMEFF2 is methylated early during oncogenesis in breast and colorectal cancer, and the detection of methylated free-circulating TMEFF2 DNA has been suggested as a potential diagnostic tool. The TMEFF2 downregulation signature equals and sometimes outperforms the Gleason and pathological scores in prostate cancer. TMEFF2 is downregulated in glioma and cotricotropinomas, and it impairs the production of adrenocorticotropic hormone in glioma cells. Interestingly, through binding the amyloid β protein, its precursor and derivatives, TMEFF2 provides neuroprotection in Alzheimer’s disease. Despite undergoing extensive investigation over the last two decades, the primary literature regarding TMEFF2 is incoherent and offers conflicting information, in particular, the oncogenic vs. onco-suppressive role of TMEFF2 in prostate cancer. For the first time, we have compiled, contextualised and critically analysed the vast body of TMEFF2-related literature and answered the apparent discrepancies regarding its function, tissue expression, intracellular localization and oncogenic vs. onco-suppressive role.

scholarly journals Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Vânia Passos ◽  
Thomas Zillinger ◽  
Nicoletta Casartelli ◽  
Amelie S. Wachs ◽  
Shuting Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Subcellular Localization ◽  
Transmembrane Protein ◽  
Type I ◽  
Accessory Gene ◽  
Protein Levels ◽  
Specificity And Sensitivity ◽  
Hiv 1

ABSTRACT When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface. IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

scholarly journals Tetraspanins TSP-12 and TSP-14 function redundantly to regulate the trafficking of the type II BMP receptor in Caenorhabditis elegans

2020 ◽  
Vol 117 (6) ◽  
pp. 2968-2977
Author(s):  
Zhiyu Liu ◽  
Herong Shi ◽  
Anthony K. Nzessi ◽  
Anne Norris ◽  
Barth D. Grant ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Expression Patterns ◽  
Bmp Signaling ◽  
Type I ◽  
Type Ii ◽  
Bmp Receptors

Tetraspanins are a unique family of 4-pass transmembrane proteins that play important roles in a variety of cell biological processes. We have previously shown that 2 paralogous tetraspanins in Caenorhabditis elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling. The underlying molecular mechanisms, however, are not fully understood. In this study, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple cell types, and that both proteins are localized on the cell surface and in various types of endosomes, including early, late, and recycling endosomes. Animals lacking both TSP-12 and TSP-14 exhibit reduced cell-surface levels of the BMP type II receptor DAF-4/BMPRII, along with impaired endosome morphology and mislocalization of DAF-4/BMPRII to late endosomes and lysosomes. These findings indicate that TSP-12 and TSP-14 are required for the recycling of DAF-4/BMPRII. Together with previous findings that the type I receptor SMA-6 is recycled via the retromer complex, our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors and highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. As TSP-12 and TSP-14 are conserved in mammals, our findings suggest that the mammalian TSP-12 and TSP-14 homologs may also function in regulating transmembrane protein recycling and BMP signaling.

scholarly journals Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Angke Zhang ◽  
Hong Duan ◽  
Huijun Zhao ◽  
Huancheng Liao ◽  
Yongkun Du ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Protein ◽  
Fluorescent Labeling ◽  
Host Cells ◽  
Respiratory Syndrome Virus ◽  
Type I ◽  
Antiviral Immune Response ◽  
Viral Membrane ◽  
Viral Membrane Fusion

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection eliminates production of type I interferons (IFNs) in host cells, which triggers an antiviral immune response through the induction of downstream IFN-stimulated genes (ISGs), thus escaping the fate of host-mediated clearance. The IFN-induced transmembrane 3 (IFITM3) has recently been identified as an ISG and plays a pivotal role against enveloped RNA viruses by restricting cell entry. However, the role of IFITM3 in PRRSV replication is unknown. The present study demonstrated that overexpression of IFITM3 suppresses PRRSV replication, while silencing of endogenous IFITM3 prominently promoted PRRSV replication. Additionally, it was shown that IFITM3 undergoes S-palmitoylation and ubiquitination modification, and both posttranslational modifications contribute to the anti-PRRSV activity of IFITM3. Further study showed that PRRSV particles are transported into endosomes and then into lysosomes during the early stages of infection, and confocal microscopy results revealed that PRRSV particles are transported to IFITM3-positive cellular vesicles. By using a single virus particle fluorescent labeling technique, we confirmed that IFITM3 can restrict PRRSV membrane fusion by inducing accumulation of cholesterol in cellular vesicles. Additionally, we found that both endogenous and exogenous IFITM3 are incorporated into newly producing PRRS virions and diminish viral intrinsic infectivity. By using cell coculture systems, we found that IFITM3 effectively restricted PRRSV intercellular transmission, which may have been caused by disrupted membrane fusion and reduced viral infectivity. In conclusion, our results demonstrate, for the first time, that swine IFITM3 interferes with the life cycle of PRRSV, and possibly other enveloped arteritis viruses, at multiple steps. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an interferon (IFN)-stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions, and this decreased their intrinsic infectivity.

scholarly journals Role of MxB in Alpha Interferon-Mediated Inhibition of HIV-1 Infection

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Bin Xu ◽  
Qinghua Pan ◽  
Chen Liang
Keyword(s):  
G Protein ◽  
Transmembrane Protein ◽  
Alpha Interferon ◽  
Wild Type Virus ◽  
Target Cells ◽  
Type I ◽  
Wide Range ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACTType I interferon inhibits viruses through inducing the expression of antiviral proteins, including the myxovirus resistance (Mx) proteins. Compared to the human MxA protein, which inhibits a wide range of viruses, the MxB protein has been reported to specifically inhibit primate lentiviruses, including HIV-1, and herpesviruses. Further, the role of endogenous MxB in alpha interferon-mediated inhibition of HIV-1 infection was questioned by a recent study showing that MxB knockout did not increase the level of infection by HIV-1 which carried the G protein of vesicular stomatitis virus (VSV), allowing infection of CD4-negative HT1080 cells. In order to further examine the anti-HIV-1 activity of endogenous MxB, we have used CRISPR/Cas9 to deplete MxB in different cell lines and observed a substantial restoration of HIV-1 infection in the presence of alpha interferon treatment. However, this rescue effect of MxB knockout became much less pronounced when infection was performed with HIV-1 carrying the VSV G protein. Interestingly, a CRISPR/Cas9 knockout screen of alpha interferon-stimulated genes in U87-MG cells revealed that the genes for interferon-induced transmembrane protein 2 (IFITM2) and IFITM3 inhibited VSV G-pseudotyped HIV-1 much more strongly than the rest of the genes tested, including the gene for MxB. Therefore, our results demonstrate the importance of MxB in alpha interferon-mediated inhibition of HIV-1 infection, which, however, can be underestimated if infection is performed with VSV G protein-pseudotyped HIV-1, due to the high sensitivity of VSV G-mediated infection to inhibition by IFITM proteins.IMPORTANCEThe results of this study reconcile the controversial reports regarding the anti-HIV-1 function of alpha interferon-induced MxB protein. In addition to the different cell types that may have contributed to the different observations, our data also suggest that VSV G protein-pseudotyped HIV-1 is much less inhibited by alpha interferon-induced MxB than HIV-1 itself is. Our results clearly demonstrate an important contribution of MxB to alpha interferon-mediated inhibition of HIV-1 in CD4+T cells, which calls for using HIV-1 target cells and wild-type virus to test the relevance of the anti-HIV-1 activity of endogenous MxB and other restriction factors.

scholarly journals Analysis of the Role of N-glycosylation in Cell-surface expression, Function and Binding Properties of SARS-CoV-2 receptor ACE2

2021 ◽  
Author(s):  
Alberto Brandariz-Nuñez ◽  
Raymond R Rowland
Keyword(s):  
Cell Surface ◽  
Viral Entry ◽  
Biological Activities ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Type I ◽  
Protein Activity ◽  
Host Cell Membrane ◽  
S Protein

Human angiotensin I-converting enzyme 2 (hACE2) is a type-I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV and SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation plays no role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell to cell fusion mediated by SARS-CoV S protein but not SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which could be the result of low cell surface expression of the deglycosylated ACE2 receptor.

Screening and Verification of Host Proteins Interacting with Neospora Caninum AMA1 Using a Yeast Two-hybrid System

2020 ◽  
Author(s):  
Baoyi Wu ◽  
Chunmei Jin ◽  
Yinan Wang ◽  
Yan Zhang ◽  
Tianyu Zheng ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Transmembrane Protein ◽  
Type I ◽  
Filamin A ◽  
Biological Functions ◽  
Yeast Two Hybrid ◽  
Apical Membrane Antigen 1 ◽  
Invasion Mechanism ◽  
First Time ◽  
Two Hybrid

Abstract Background: Neospora caninum apical membrane antigen 1 (NcAMA1) is a conservative type I transmembrane protein that is secreted by the microneme to the surface of the parasite, and is a key component of the invasion mechanism. In order to explore further the biological functions of NcAMA1 in the process of parasite invasion, we conducted research on NcAMA1 and its interacting partners. Methods: In this study, Vero cell yeast two-hybrid (Y2H) cDNA library was constructed. Using the constructed recombinant vector pGBKT7-NcAMA1 as bait, the Y2H system was used to screen the proteins that interact with NcAMA1. In addition, the interaction between NcAMA1 and the screened transmembrane emp24 domain trafficking protein 2 （Tmed2）was further verified by one-to-one Y2H experiments and pull-down, and the role of Tmed2 protein in the process of N. caninum invasion was initially verified by RNA silencing and antibody blocking experiments.Results: Our results show that, through the Y2H experiment, we have identified two proteins that interact with NcAMA1, which are the Chlorocebus sabaeus filamin A, alpha and Chlorocebus sabaeus Tmed2. When the expression of Tmed2 protein decreased or blocked, the invasion rate of N. caninum was increased.Conclusions: These findings give us a deeper understanding of the biological functions of NcAMA1, and for the first time suggest that Tmed2 may be involved in the process invasion by of N. caninum, inhibiting the invasion of parasites by interacting with the protein secreted by N. caninum.

scholarly journals Membrane-anchored growth factor, HB-EGF, on the cell surface targeted to the inner nuclear membrane

2008 ◽  
Vol 180 (4) ◽  
pp. 763-769 ◽  
Author(s):  
Miki Hieda ◽  
Mayumi Isokane ◽  
Michiko Koizumi ◽  
Chiduru Higashi ◽  
Taro Tachibana ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Transmembrane Protein ◽  
Active Form ◽  
Type I ◽  
Transcriptional Repressors ◽  
Sequence Element ◽  
Inner Nuclear Membrane

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

Functional insights into the role of novel type I cohesin and dockerin domains from Clostridium thermocellum

2009 ◽  
Vol 424 (3) ◽  
pp. 375-384 ◽  
Author(s):  
Benedita A. Pinheiro ◽  
Harry J. Gilbert ◽  
Kazutaka Sakka ◽  
Kazuo Sakka ◽  
Vânia O. Fernandes ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Type I ◽  
Cell Wall Polysaccharides ◽  
Molecular Scaffold ◽  
Catalytic Subunits ◽  
Anaerobic Microorganisms ◽  
Binding Interfaces

Cellulosomes, synthesized by anaerobic microorganisms such as Clostridium thermocellum, are remarkably complex nanomachines that efficiently degrade plant cell wall polysaccharides. Cellulosome assembly results from the interaction of type I dockerin domains, present on the catalytic subunits, and the cohesin domains of a large non-catalytic integrating protein that acts as a molecular scaffold. In general, type I dockerins contain two distinct cohesin-binding interfaces that appear to display identical ligand specificities. Inspection of the C. thermocellum genome reveals 72 dockerin-containing proteins. In four of these proteins, Cthe_0258, Cthe_0435, Cthe_0624 and Cthe_0918, there are significant differences in the residues that comprise the two cohesin-binding sites of the type I dockerin domains. In addition, a protein of unknown function (Cthe_0452), containing a C-terminal cohesin highly similar to the equivalent domains present in C. thermocellum-integrating protein (CipA), was also identified. In the present study, the ligand specificities of the newly identified cohesin and dockerin domains are described. The results revealed that Cthe_0452 is located at the C. thermocellum cell surface and thus the protein was renamed as OlpC. The dockerins of Cthe_0258 and Cthe_0435 recognize, preferentially, the OlpC cohesin and thus these enzymes are believed to be predominantly located at the surface of the bacterium. By contrast, the dockerin domains of Cthe_0624 and Cthe_0918 are primarily cellulosomal since they bind preferentially to the cohesins of CipA. OlpC, which is a relatively abundant protein, may also adopt a ‘warehouse’ function by transiently retaining cellulosomal enzymes at the cell surface before they are assembled on to the multienzyme complex.

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