GSDM family genes meet autophagy

2015 ◽  
Vol 469 (2) ◽  
pp. e5-e7 ◽  
Author(s):  
Masaru Tamura ◽  
Toshihiko Shiroishi
Keyword(s):  
Protein Function ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Autophagic Activity

Shi et al. provided a new insight of Gsdm family protein function. They reported that N- and C-terminus domain of Gsdm family protein interact each other. Moreover, the N-terminus domain of Gsdm family proteins has pro-autophagic activity, which is suppressed by the C-terminus domain.

scholarly journals Maturation of active zone assembly by Drosophila Bruchpilot

2009 ◽  
Vol 186 (1) ◽  
pp. 129-145 ◽  
Author(s):  
Wernher Fouquet ◽  
David Owald ◽  
Carolin Wichmann ◽  
Sara Mertel ◽  
Harald Depner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drosophila Melanogaster ◽  
High Resolution ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Dense Projections ◽  
Ca2 Channels

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

scholarly journals Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity

2013 ◽  
Vol 203 (2) ◽  
pp. 299-313 ◽  
Author(s):  
Yasuhiro Araki ◽  
Wei-Chi Ku ◽  
Manami Akioka ◽  
Alexander I. May ◽  
Yu Hayashi ◽  
...  
Keyword(s):  
Complex I ◽  
Protein Complexes ◽  
Pi3 Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
C Terminus ◽  
Physical Linkage ◽  
N Terminus ◽  
Autophagic Activity ◽  
Biochemical Analyses ◽  
Phosphatidylinositol 3

Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.

scholarly journals Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

2021 ◽  
Vol 22 (11) ◽  
pp. 5709
Author(s):  
Mitzi Díaz-Hernández ◽  
Rosario Javier-Reyna ◽  
Izaid Sotto-Ortega ◽  
Guillermina García-Rivera ◽  
Sarita Montaño ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Entamoeba Histolytica ◽  
Posttranslational Modifications ◽  
Target Protein ◽  
Binding Motif ◽  
Docking Analysis ◽  
Membrane Activity ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

scholarly journals Cotranslational assembly imposes evolutionary constraints on homomeric proteins

2016 ◽  
Author(s):  
Eviatar Natan ◽  
Tamaki Endoh ◽  
Liora Haim-Vilmovsky ◽  
Guilhem Chalancon ◽  
Tilman Flock ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Complexes ◽  
Evolutionary Constraints ◽  
E Coli ◽  
Protein Library ◽  
C Terminus ◽  
N Terminus ◽  
Polypeptide Chains

AbstractThere is increasing evidence that some proteins fold during translation, i.e. cotranslationally, which implies that partial protein function, including interactions with other molecules, could potentially be unleashed early on during translation. Although little is known about cotranslational assembly mechanisms, for homomeric protein complexes, translation by the ribosome, folding and assembly, should be well-coordinated to avoid misassembly in the context of polysomes. We analysed 3D structures of homomers and identified a statistically significant trend conserved across evolution that supports this hypothesis: namely that homomeric contacts tend to be localized towards the C-terminus rather than N-terminus of homomeric polypeptide chains. To probe this in more detail, we expressed a GFP-based library of 611 homomeric E. coli genes, and analyzing their folding and assembly in vivo. Consistent with our hypothesis, interface residues tend to be located near the N-terminus in cotranslationally aggregating homomers. In order to dissect the mechanisms of folding and assembly under controlled conditions, we engineered a protein library with three variable components: (i) the position and type homomerization domain, (ii) the reporter domain and (iii) the linker length that connects the two. By analyzing the misassembly rates of these engineered constructs in vivo, in vitro and in silico, we confirmed our hypothesis that C-terminal homomerization is favorable to N-terminal homomerization. More generally, these results provide a set of spatiotemporal constraints within polypeptide chains that favor efficient assembly, with implications for protein evolution and design.

Preparation and properties of three analogues of [5-leucine]enkephalin, substrates for enzyme conversion studies

1985 ◽  
Vol 50 (6) ◽  
pp. 1329-1334
Author(s):  
Jaroslav Vičar ◽  
Linda Servítová ◽  
Martin Flegel ◽  
Karel Hauzer ◽  
Tomislav Barth
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Guinea Pig ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Guinea Pig Ileum ◽  
Opioid Activity ◽  
C Terminus ◽  
N Terminus ◽  
Fragment Condensation

Analogues of [5-Leu]enkephalin, prolonged by methionine on the N-terminus or, by lysine or methionine on the C-terminus were prepared by fragment condensation, purified by ion exchange chromatography or high-pressure liquid chromatography. The substances were characterised by their opioid activity in a test on guinea-pig ileum in comparison with the activity of [5-Leu]enkephalin.

scholarly journals Improved Antimicrobial Activities of Synthetic-Hybrid Bacteriocins Designed from Enterocin E50-52 and Pediocin PA-1

2014 ◽  
Vol 81 (5) ◽  
pp. 1661-1667 ◽  
Author(s):  
Santosh Kumar Tiwari ◽  
Katia Sutyak Noll ◽  
Veronica L. Cavera ◽  
Michael L. Chikindas
Keyword(s):  
Micrococcus Luteus ◽  
Electrical Potential ◽  
Gram Negative Bacteria ◽  
Additional Advantage ◽  
Gram Negative ◽  
Content Type ◽  
Intracellular Atp ◽  
Hybrid Peptides ◽  
C Terminus ◽  
N Terminus

ABSTRACTTwo hybrid bacteriocins, enterocin E50-52/pediocin PA-1 (EP) and pediocin PA-1/enterocin E50-52 (PE), were designed by combining the N terminus of enterocin E50-52 and the C terminus of pediocin PA-1 and by combining the C terminus of pediocin PA-1 and the N terminus of enterocin E50-52, respectively. Both hybrid bacteriocins showed reduced MICs compared to those of their natural counterparts. The MICs of hybrid PE and EP were 64- and 32-fold lower, respectively, than the MIC of pediocin PA-1 and 8- and 4-fold lower, respectively, than the MIC of enterocin E50-52. In this study, the effect of hybrid as well as wild-type (WT) bacteriocins on the transmembrane electrical potential (ΔΨ) and their ability to induce the efflux of intracellular ATP were investigated. Enterocin E50-52, pediocin PA-1, and hybrid bacteriocin PE were able to dissipate ΔΨ, but EP was unable to deplete this component. Both hybrid bacteriocins caused a loss of the intracellular concentration of ATP. EP, however, caused a faster efflux than PE and enterocin E50-52. Enterocin E50-52 and hybrids PE and EP were active against the Gram-positive and Gram-negative bacteria tested, such asMicrococcus luteus,Salmonella entericaserovar Enteritidis 20E1090, andEscherichia coliO157:H7. The hybrid bacteriocins designed and described herein are antimicrobial peptides with MICs lower those of their natural counterparts. Both hybrid peptides induce the loss of intracellular ATP and are capable of inhibiting Gram-negative bacteria, and PE dissipates the electrical potential. In this study, the MIC of hybrid bacteriocin PE decreased 64-fold compared to the MIC of its natural peptide counterpart, pediocin PA-1. Inhibition of Gram-negative pathogens confers an additional advantage for the application of these peptides in therapeutics.

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2055-2068 ◽  
Author(s):  
Daniel V. Zurawski ◽  
Murry A. Stein
Keyword(s):  
Type Iii Secretion ◽  
Coiled Coil ◽  
Amphipathic Helix ◽  
Yeast Two Hybrid ◽  
C Terminus ◽  
N Terminus ◽  
Domain Mapping ◽  
Self Association ◽  
Discrete Domains ◽  
Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

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