scholarly journals A post-translational modification of the sheath modulatesFrancisellatype VI secretion system assembly and function

2018 ◽  
Author(s):  
Jason Ziveri ◽  
Cerina Chhuon ◽  
Anne Jamet ◽  
Guénolé Prigent ◽  
Héloïse Rytter ◽  
...  
Keyword(s):  
Critical Role ◽  
Phosphorylation Site ◽  
Post Translational Modification ◽  
Secretion Systems ◽  
Post Translational Modifications ◽  
Type Vi Secretion ◽  
Sheath Formation ◽  
Canonical Type ◽  
The Stability ◽  
And Function

AbstractFrancisella tularensisis a facultative intracellular pathogen that causes the zoonotic disease tularemia in human and animal hosts. This bacterium possesses a non-canonical type VI secretion systems (T6SS) required for phagosomal escape and access to its replicative niche in the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that the amounts of essentially all the TSS6 proteins remained unchanged upon KCl stimulation. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. A whole cell phosphoproteomic analysis allowed us to identify a unique phosphorylation site on IglB, the TssC homologue and key component of the T6SS sheath. Importantly, the phosphorylated form of IglB was not present in the contracted sheath and 3D modeling indicated that the charge repulsion provoked by addition of a phosphogroup on tyrosine 139 was likely to weaken the stability of the sheath structure. Substitutions of the phosphorylatable residue of IglB (tyrosine 139) with alanine or with phosphomimetics prevented T6SS formation and totally impaired phagosomal escape. In contrast, the substitution with the non-phosphorylatable aromatic analog phenylalanine impaired but did not prevent phagosomal escape and cytosolic bacterial multiplication in J774-1 macrophages. Altogether these data suggest that phosphorylation of the sheath participates to T6SS disassembly. Post-translational modifications of the sheath may represent a previously unrecognized mechanism to finely modulate the dynamics of T6SS assembly-disassembly.Data are available via ProteomeXchange with identifier PXD012507.SynopsisFrancisellapossesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that KCl stimulation did not result in an increased production of TSS6 proteins. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. Using a global and site-specific phosphoproteomic analysis ofFrancisellawe identified a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. We show that this site plays a critical role in T6SS biogenesis and propose that phosphorylation may represent a new mechanism affecting the dynamics of sheath formation.

scholarly journals Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

2019 ◽  
Vol 18 (12) ◽  
pp. 2418-2432 ◽  
Author(s):  
Jason Ziveri ◽  
Cerina Chhuon ◽  
Anne Jamet ◽  
Héloïse Rytter ◽  
Guénolé Prigent ◽  
...  
Keyword(s):  
Critical Role ◽  
Phosphorylation Site ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
And Function

scholarly journals iProteinDB: an integrative database of Drosophila post-translational modifications

2018 ◽  
Author(s):  
Yanhui Hu ◽  
Richelle Sopko ◽  
Verena Chung ◽  
Romain A. Studer ◽  
Sean D. Landry ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Large Scale ◽  
Model Organisms ◽  
General Strategy ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Sites ◽  
Evolutionarily Conserved ◽  
And Function

AbstractPost-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing stability, protein interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, commonly serine, threonine and tyrosine. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that many phosphorylation sites may be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites with regards to regulation and function. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus laevis, Danio rerio, and Caenorhabditis elegans.

scholarly journals Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

2009 ◽  
Vol 191 (9) ◽  
pp. 2985-2992 ◽  
Author(s):  
Gisèle Bourg ◽  
Romain Sube ◽  
David O'Callaghan ◽  
Gilles Patey
Keyword(s):  
Critical Role ◽  
Type Iv Secretion ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iv ◽  
Brucella Suis ◽  
Periplasmic Domain ◽  
Tm Domain ◽  
And Function

ABSTRACT The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Pax8 protein stability is controlled by sumoylation

2008 ◽  
Vol 42 (1) ◽  
pp. 35-46 ◽  
Author(s):  
Tiziana de Cristofaro ◽  
Anna Mascia ◽  
Andrea Pappalardo ◽  
Barbara D'Andrea ◽  
Lucio Nitsch ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Critical Role ◽  
Nuclear Bodies ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Signal Transducers ◽  
Protein Protein Interaction ◽  
Sumo Ligase ◽  
Development And Differentiation ◽  
Substitution Mutant

The transcription factor Pax8 is involved in the morphogenesis of the thyroid gland and in the maintenance of the differentiated thyroid phenotype. Despite the critical role played by Pax8 during thyroid development and differentiation, very little is known of its post-translational modifications and how these modifications may regulate its activity. We focused our attention on the study of a specific post-translational modification, i.e., sumoylation. Sumoylation is a dynamic and reversible process regulating gene expression by altering transcription factor stability, protein–protein interaction and subcellular localization of target proteins. The analysis of Pax8 protein sequence revealed the presence of one sumoylation consensus motif (ψKxE), strongly conserved among mammals, amphibians, and fish. We demonstrated that Pax8 is sumoylated by the addition of a single small ubiquitin-like modifier (SUMO) molecule on its lysine residue 309 and that Pax8K309R, a substitution mutant in which the candidate lysine is replaced with an arginine, is no longer modified by SUMO. In addition, we analyzed whether protein inhibitor of activated signal transducers and activators of transcription (PIASy), a member of the PIAS STAT family of proteins, could function as a SUMO ligase and we demonstrated that indeed PIASy is able to increase the fraction of sumoylated Pax8. Interestingly, we show that Pax8 is targeted in the SUMO nuclear bodies, which are structures that regulate the nucleoplasmic concentration of transcription factors by SUMO trapping. Finally, we report here that the steady-state protein level of Pax8 is controlled by sumoylation.

scholarly journals Inborn errors E778K and G908R in NOD2 gene increase risk of nontuberculous mycobacterial infection: a computational study

2020 ◽  
Author(s):  
Shamila D. Alipoor ◽  
Mehdi Mirsaeidi
Keyword(s):  
Computational Study ◽  
Critical Role ◽  
Mycobacterial Infection ◽  
Downstream Processing ◽  
Post Translational Modification ◽  
Inborn Errors ◽  
Increase Risk ◽  
Receptor Crosstalk ◽  
Nontuberculous Mycobacterial Infection ◽  
And Function

AbstractBackgroundThe innate immune system has a critical role in the early detection of pathogens, mainly by relying on pattern-recognition receptor (PRR) signaling molecules. Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic sensor for recognition of invading molecules and danger signals inside the cells. NOD2’s functions are critical; polymorphisms of its encoding gene are associated with several immune pathological conditions. We recently reported that missense E778K and G908R variants of NOD2 gene are associated with recurrent pulmonary nontuberculous mycobacterial infectionsMethodsThis is an in-silico analysis of NOD2 gene using SNPs functionality analyses, post-translational modification site prediction and network analysis.ResultsOur analysis revealed that these damaging mutations affect the structural properties and function and ligand binding in the mutant receptor.ConclusionThe consequence of these mutations may also impress downstream processing and receptor crosstalk with other immune molecules and therefore increase susceptibility to infectious disease.

scholarly journals Regulation of poly(a)-specific ribonuclease activity by reversible lysine acetylation

2020 ◽  
Vol 295 (30) ◽  
pp. 10255-10270
Author(s):  
Eden A. Dejene ◽  
Yixuan Li ◽  
Zahra Showkatian ◽  
Hongbo Ling ◽  
Edward Seto
Keyword(s):  
Enzymatic Activity ◽  
Noncoding Rna ◽  
Critical Role ◽  
Telomere Maintenance ◽  
Post Translational Modifications ◽  
Telomere Elongation ◽  
The Stability ◽  
Human Telomerase

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3′-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3′-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.

scholarly journals Phosphorylation-Induced Ubiquitination and Degradation of PXR through CDK2-TRIM21 Axis

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 264
Author(s):  
Mengyao Qin ◽  
Yu Xin ◽  
Yong Bian ◽  
Xuan Yang ◽  
Tao Xi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Phosphorylation Site ◽  
Pregnane X Receptor ◽  
Cdk Inhibitors ◽  
Post Translational Modification ◽  
Downstream Target ◽  
Primary Mouse ◽  
Wide Range ◽  
The Stability

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that is activated by a variety of endogenous metabolites or xenobiotics. Its downstream target genes are involved in metabolism, inflammation and processes closely related to cancer. However, the stability regulation of PXR protein resulting from post-translational modification is still largely undefined. In the present study, primary mouse hepatocytes, hepatoma HepG2 cells and HEK 293T cells were used to investigate gene expression and protein interactions. The role of kinases was evaluated by RNA interference and overexpression constructs with or without PXR phosphorylation site mutations. The activity of CYP3A4 and P-gp was determined by enzymatic and substrate accumulation assays. It was found that E3 ubiquitin ligase TRIM21 mediates the ubiquitination and degradation of PXR and plays an important role in regulating the activity of PXR. On this basis, PXR phosphorylation-associated kinases were evaluated regarding regulation of the stability of PXR. We found cyclin dependent kinase 2 (CDK2) exclusively phosphorylates PXR at Ser350, promotes its disassociation with Hsp90/DNAJC7, and leads to subsequent TRIM21-mediated PXR ubiquitination and degradation. As well-known CDK inhibitors, dinaciclib and kenpaullone stabilize PXR and result in elevated expression and activity of PXR-targeted DMETs, including carboxylesterases, CYP3A4 and P-gp. The suppressed degradation of PXR by CDK2 inhibitors denotes dinaciclib-induced promotion of PXR-targeted genes. The findings of CDK2-mediated PXR degradation indicate a wide range of potential drug–drug interactions during clinical cancer therapy using CDK inhibitors and imply an alternative direction for the development of novel PXR antagonists.

scholarly journals The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ahmad Abdelzaher Zaki Khalifa ◽  
Muneyoshi Ichikawa ◽  
Daniel Dai ◽  
Shintaroh Kubo ◽  
Corbin Steven Black ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tetrahymena Thermophila ◽  
Building Blocks ◽  
Post Translational Modifications ◽  
Ciliary Beating ◽  
Cryo Electron Microscopy ◽  
The Stability ◽  
And Function ◽  
Β Tubulin ◽  
Junction Proteins

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

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