scholarly journals A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 902 ◽  
Author(s):  
Steve Catarino ◽  
Teresa M Ribeiro-Rodrigues ◽  
Rita Sá Ferreira ◽  
José Ramalho ◽  
Christine Abert ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Protein Interaction ◽  
Alternative Mechanism ◽  
Direct Communication ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Protein Ubiquitination ◽  
Lir Motif ◽  
Vitro Protein

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report, we describe a functional LC3 interacting region (LIR), present in the amino terminal of most Cx protein family members, which can mediate the autophagy degradation of Cx43 without the need of ubiquitin. Mutation of the LIR motif on Cx37, Cx43, Cx46 and Cx50 impairs interaction with LC3B and GABARAP without compromising protein ubiquitination. Through in vitro protein-protein interaction assays, we demonstrate that this LIR motif is required for the binding of Cx43 to LC3B and GABARAP. Overall, our findings describe an alternative mechanism whereby Cxs interact with LC3/GABARAP proteins, envisioning a new model for the autophagy degradation of connexins.

scholarly journals Prdx1 Interacts with ASK1 upon Exposure to H2O2 and Independently of a Scaffolding Protein

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1060
Author(s):  
Trung Nghia Vo ◽  
Julia Malo Pueyo ◽  
Khadija Wahni ◽  
Daria Ezeriņa ◽  
Jesalyn Bolduc ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Interaction ◽  
Specific Protein ◽  
Annexin A2 ◽  
Redox Signaling ◽  
Scaffolding Protein ◽  
Protein Protein Interaction ◽  
Protein Thiols ◽  
Vitro Protein

Hydrogen peroxide (H2O2) is a key redox signaling molecule that selectively oxidizes cysteines on proteins. It can accomplish this even in the presence of highly efficient and abundant H2O2 scavengers, peroxiredoxins (Prdxs), as it is the Prdxs themselves that transfer oxidative equivalents to specific protein thiols on target proteins via their redox-relay functionality. The first evidence of a mammalian cytosolic Prdx-mediated redox-relay—Prdx1 with the kinase ASK1—was presented a decade ago based on the outcome of a co-immunoprecipitation experiment. A second such redox-relay—Prdx2:STAT3—soon followed, for which further studies provided insights into its specificity, organization, and mechanism. The Prdx1:ASK1 redox-relay, however, has never undergone such a characterization. Here, we combine cellular and in vitro protein–protein interaction methods to investigate the Prdx1:ASK1 interaction more thoroughly. We show that, contrary to the Prdx2:STAT3 redox-relay, Prdx1 interacts with ASK1 at elevated H2O2 concentrations, and that this interaction can happen independently of a scaffolding protein. We also provide evidence of a Prdx2:ASK1 interaction, and demonstrate that it requires a facilitator that, however, is not annexin A2. Our results reveal that cytosolic Prdx redox-relays can be organized in different ways and yet again highlight the differentiated roles of Prdx1 and Prdx2.

scholarly journals Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

2007 ◽  
Vol 88 (12) ◽  
pp. 3270-3274 ◽  
Author(s):  
Marianne Bonvin ◽  
Jobst Greeve
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Amino Terminal ◽  
Hbv Replication ◽  
B Virus ◽  
Carboxy Terminal ◽  
Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin

2017 ◽  
Author(s):  
Carolina dos S. Passos ◽  
Nathalie Deschamps ◽  
Yun Choi ◽  
Robert E. Cohen ◽  
Remo Perozzo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Stress Response ◽  
Small Molecules ◽  
Histone Deacetylase ◽  
Protein Interaction ◽  
Potential Role ◽  
Histone Deacetylase 6 ◽  
Protein Protein Interaction ◽  
Binding Function

AbstractHistone deacetylase 6 (HDAC6) is a cytoplasmic HDAC isoform able to remove acetyl groups from cellular substrates such as α-tubulin. In addition to the two deacetylase domains, HDAC6 has a C-terminal zinc-finger ubiquitin (Ub)-binding domain (ZnF-UBP) able to recognize free Ub. HDAC6-Ub interaction is thought to function in regulating the elimination of misfolded proteins during stress response through the aggresome pathway. Small molecules inhibiting deacetylation by HDAC6 were shown to reduce aggresomes, but the interplay between HDAC6 catalytic activity and Ub-binding function is not fully understood. Here we describe two methods to measure the HDAC6-Ub interaction in vitro using full-length HDAC6. Both methods were effective for screening inhibitors of the HDAC6-Ub protein-protein interaction independently of the catalytic activity. Our results suggest a potential role for the HDAC6 deacetylase domains in modulating HDAC6-Ub interaction. This new aspect of HDAC6 regulation can be targeted to address the roles of HDAC6-Ub interaction in normal and disease conditions.

Specificity of protein-RNA and protein-protein interaction upon assembly of TMV in vivo and in vitro

Virology ◽  
1975 ◽  
Vol 67 (1) ◽  
pp. 1-13 ◽  
Author(s):  
T.I. Atabekova ◽  
M.E. Taliansky ◽  
J.G. Atabekov
Keyword(s):  
Protein Interaction ◽  
Protein Protein Interaction

scholarly journals Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions

2002 ◽  
Vol 365 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Translation System ◽  
Alternative Mechanism ◽  
Protein Cleavage ◽  
Membrane Components ◽  
Gap Junction Hemichannels

Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.

scholarly journals Evolutionary diversification of protein–protein interactions by interface add-ons

2017 ◽  
Vol 114 (40) ◽  
pp. E8333-E8342 ◽  
Author(s):  
Maximilian G. Plach ◽  
Florian Semmelmann ◽  
Florian Busch ◽  
Markus Busch ◽  
Leonhard Heizinger ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Evolutionary Strategy ◽  
Structural Level ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

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