scholarly journals Overexpression of the Selective Autophagy Cargo Receptor NBR1 Modifies Plant Response to Sulfur Deficit

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 669 ◽  
Author(s):  
Leszek Tarnowski ◽  
Milagros Collados Rodriguez ◽  
Jerzy Brzywczy ◽  
Dominik Cysewski ◽  
Anna Wawrzynska ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Kinase Inhibitors ◽  
Biological Significance ◽  
Direct Interaction ◽  
Ribosomal Gene ◽  
Plant Response ◽  
Wild Type ◽  
Selective Autophagy ◽  
Cargo Receptor

Plants exposed to sulfur deficit elevate the transcription of NBR1 what might reflect an increased demand for NBR1 in such conditions. Therefore, we investigated the role of this selective autophagy cargo receptor in plant response to sulfur deficit (-S). Transcriptome analysis of the wild type and NBR1 overexpressing plants pointed out differences in gene expression in response to -S. Our attention focused particularly on the genes upregulated by -S in roots of both lines because of significant overrepresentation of cytoplasmic ribosomal gene family. Moreover, we noticed overrepresentation of the same family in the set of proteins co-purifying with NBR1 in -S. One of these ribosomal proteins, RPS6 was chosen for verification of its direct interaction with NBR1 and proven to bind outside the NBR1 ubiquitin binding domains. The biological significance of this novel interaction and the postulated role of NBR1 in ribosomes remodeling in response to starvation remain to be further investigated. Interestingly, NBR1 overexpressing seedlings have significantly shorter roots than wild type when grown in nutrient deficient conditions in the presence of TOR kinase inhibitors. This phenotype probably results from excessive autophagy induction by the additive effect of NBR1 overexpression, starvation, and TOR inhibition.

scholarly journals NBR1 directly promotes the formation of p62 – ubiquitin condensates via its PB1 and UBA domains

2020 ◽  
Author(s):  
Adriana Savova ◽  
Julia Romanov ◽  
Sascha Martens
Keyword(s):  
Phase Separation ◽  
Wild Type ◽  
Selective Autophagy ◽  
Ubiquitinated Proteins ◽  
Cargo Receptor ◽  
Condensate Formation ◽  
Uba Domain ◽  
Reconstituted System ◽  
Pb1 Domain

SummarySelective autophagy removes harmful intracellular structures such as ubiquitinated, aggregated proteins ensuring cellular homeostasis. This is achieved by the encapsulation of this cargo material within autophagosomes. The cargo receptor p62/SQSTM1 mediates the phase separation of ubiquitinated proteins into condensates, which subsequently become targets for the autophagy machinery. NBR1, another cargo receptor, is a crucial regulator of condensate formation. The mechanisms of the interplay between p62 and NBR1 are not well understood. Employing a fully reconstituted system we show that two domains of NBR1, the PB1 domain which binds to p62 and the UBA domain which binds to ubiquitin, are required to promote p62-ubiquitin condensate formation. In cells, acute depletion of endogenous NBR1 reduces formation of p62 condensates, a phenotype that can be rescued by re-expression of wild-type NBR1, but not PB1 or UBA domain mutants. Our results provide mechanistic insights into the role of NBR1 in selective autophagy.

scholarly journals Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
You-Chul Jung ◽  
Mi-Ae Lee ◽  
Kyu-Ho Lee
Keyword(s):  
Biofilm Formation ◽  
Specific Binding ◽  
Biological Significance ◽  
Wild Type ◽  
Forming Process ◽  
Homologous Proteins ◽  
Content Type ◽  
Pathogenic Vibrio ◽  
Biofilm Matrix

ABSTRACT The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation. IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

scholarly journals The dynamic switch mechanism that leads to activation of LRRK2 is embedded in the DFGψ motif in the kinase domain

2019 ◽  
Vol 116 (30) ◽  
pp. 14979-14988 ◽  
Author(s):  
Sven H. Schmidt ◽  
Matthias J. Knape ◽  
Daniela Boassa ◽  
Natascha Mumdey ◽  
Alexandr P. Kornev ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Regulatory Mechanism ◽  
Kinase Domain ◽  
Leucine Rich Repeat ◽  
Wild Type ◽  
Conformational Switch ◽  
Multidomain Protein ◽  
Switch Mechanism ◽  
And Function

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson’s disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a “brake” that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.

scholarly journals Nα-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Ulrike A. Friedrich ◽  
Mostafa Zedan ◽  
Bernd Hessling ◽  
Kai Fenzl ◽  
Ludovic Gillet ◽  
...  
Keyword(s):  
Stress Responses ◽  
Ribosomal Proteins ◽  
Protein Modification ◽  
Protein Quality ◽  
Biological Significance ◽  
General Role ◽  
Altered Expression ◽  
Phenotypic Differences ◽  
Genes Encoding

SummaryN-terminal (Nt)-acetylation is a highly prevalent co-translational protein modification in eukaryotes, catalyzed by at least five Nt-acetyltransferases (Nat) with differing specificities. Nt-acetylation has been implicated in protein quality control but its broad biological significance remains elusive. We investigated the roles of the two major Nats of S. cerevisiae, NatA and NatB, by performing transcriptome, translatome and proteome profiling of natAΔ and natBΔ mutants. Our results do not support a general role of Nt-acetylation in protein degradation but reveal an unexpected range of Nat-specific phenotypes. NatA is implicated in systemic adaptation control, as natAΔ mutants display altered expression of transposons, sub-telomeric genes, pheromone response genes and nuclear genes encoding mitochondrial ribosomal proteins. NatB predominantly affects protein folding, as natBΔ mutants accumulate protein aggregates, induce stress responses and display reduced fitness in absence of the ribosome-associated chaperone Ssb. These phenotypic differences indicate that controlling Nat activities may serve to elicit distinct cellular responses.

scholarly journals The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 213-213
Author(s):  
Shabbir Ansari ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Specific Activity ◽  
Direct Interaction ◽  
Lipid Binding ◽  
Wild Type ◽  
Binding Region ◽  
Coagulant Activity ◽  
In Cells

Abstract Tissue factor (TF) is the cellular cofactor for the serine protease coagulation factor VIIa (FVIIa). The TF-FVIIa complex formed on the cell surface initiates the coagulation cascade. It is believed that most of the TF molecules on the cell surface of a resting cell exist in an encrypted state with very little procoagulant activity. Encrypted TF must undergo decryption to become fully active. The exact mechanisms by which TF activity on the cell surface is regulated are unknown. Exposure of phosphatidylserine (PS) to the outer leaflet of the cell membrane is thought to play a critical role in TF decryption. Recent studies of molecular dynamics simulation of TF ectodomain in solution and on the surface of anionic phospholipids suggested a direct interaction of PS headgroups with specific residues in TF. At present, the role of the putative lipid interactive residues of TF in TF decryption is unknown. In the present study, we investigated the potential role of TF direct interaction with the cell surface lipids on basal TF activity as well as enhanced TF activity following the decryption using different TF mutants. Plasmids or adenoviral constructs encoding wild-type or mutant TF (mutations in the putative lipid binding region) were used to transduce TF expression in CHO-K1 or monocytic THP-1 cells, respectively. TF protein expression level at the cell surface and FVIIa binding to the cell surface TF were evaluated by radioligand binding studies using 125I-labeled TF mAb or FVIIa, respectively. TF-FVIIa coagulant activity on the cell surface was determined in FX activation assay. Data of these studies showed that all TF mutants were capable of interacting with FVIIa with no apparent defect. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A-exhibited a similar or slightly higher TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A and TFK166A, was reduced substantially to a range of 40% - 70% of that of wild-type TF. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% loss of TF specific activity. Mutation of all nine lipid binding residues together (DLBR) did not further decrease the specific activity of TF anymore than that of mutation of G164 alone. Comparison of the present data with the published data on these mutants revealed that some of the TF residues that are critical for regulating TF activity on liposomes are not crucial for TF activity on the cell surface. To address whether the decreased FXa generation seen with the select TF variants is caused by changes in TF-membrane interaction or by the substrate interaction with TF/FVIIa complex, we performed Michaelis-Menten kinetics of FX activation for two of TF mutants (TFS163A and TFG164A). Results of this study suggested that there were no significant differences in Km values between wild-type TF and TF mutants (wild-type TF, 51 ± 14.6 nM; TFS163A, 68 ± 19.5 nM; TFG164A, 39 ± 18.4 nM, n=4). Interestingly, mutation of the selective residues in the lipid binding region failed to abrogate the PS-dependent TF decryption. The fold-increase in TF activity in cells expressing wild-type TF or TF variants was similar following cell activation with either HgCl2 or calcium ionomycin treatment. Annexin V markedly diminished the increased TF-FVIIa activation of FX in cells expressing wild-type TF as well as cells expressing the TF mutant (DLBR mutant). Overall, our data suggest that the regulation of TF activity at the cell surface milieu may be different from that of PC/PS vesicles and TF region other than earlier identified LBR may be responsible for enhancing TF activity following the PS exposure. Disclosures No relevant conflicts of interest to declare.

Downregulation of Ribosomal Proteins Is Seen in Non 5q- MDS

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 854-854 ◽  
Author(s):  
Davendra P. Sohal ◽  
Andrea Pellagati ◽  
Li Zhou ◽  
Yongkai Mo ◽  
Joanna B. Opalinska ◽  
...  
Keyword(s):  
High Resolution ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Expression Profiles ◽  
Ribosomal Gene ◽  
Comparative Genome Hybridization ◽  
Gene Promoters ◽  
Genomic Deletions ◽  
Cd34 Cells

Abstract Recent work (Ebert et al, Nature 2008, Jan 17:335-9) has shown that ribosomal protein S14 (RPS14) is underexpressed in myelodysplasia (MDS) with deletion of chromosome 5q and plays a role in its pathogenesis. The role of ribosomal proteins in other more common subtypes of MDS is unknown. We conducted a meta-analytical comparison of gene expression profiles of 60 cases of non 5q- MDS CD34+ cells with 52 normal CD34+ profiles. These datasets were obtained from seven independent studies from NCBI’s GEO database. The data was integrated based on UniGene IDs and were quantile normalized to ensure cross-study comparability. (Based on our previous approach; Sohal et al PLOS One, 2008, Zhou et al, Blood, 2008). Using significance analysis of microarrays (SAM) and a false discovery rate (FDR) of just 0.04%, we found that ribosomal protein were the class of genes that were most significantly altered in MDS. We observed that RPL35a, RPS9, RPL10, RPL22, RPS14, RPS10, RPS15a, RPS24, RPL24, RPL36, RPL21, RPL23 were strikingly downregulated in non-5q- MDS CD34+ cells. To determine if these alterations were a result of changes in DNA copy numbers of these genes, we examined 20 MDS samples by high resolution array comparative genome hybridization (aCGH) performed on Nimblegen whole genome tiling arrays. aCGH at 6kb resolution revealed deletions in RPL14, RPL22, RPL36, RPS10, RPS5 and even RPS14 in distinct selected cases of non 5q- MDS. These small deletions, which were not identifiable by traditional karyotyping methods, may be putative mechanisms of ribosomal protein downregulation and ultimately, in MDS development and/or progression. Since MDS is also characterized by aberrant epigenetic silencing of genes, we next examined the methylation status of ribosomal gene promoters by high resolution global DNA methylation profiling by using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)). This assay uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. While there were sporadic changes in some patients, we did not observe any consistently significant changes in methylation of these ribosomal gene promoters on comparison with normal anemic controls. In summary, we show novel widespread alterations in ribosomal protein expression in MDS, at least some of which are associated with genomic deletions. These findings illustrate the applicability of meta-analytical genomic approaches in a heterogeneous disease such as MDS. Most importantly, our data points to the dramatic role of alteration in the protein translational machinery in pathogenesis of MDS.

scholarly journals Heterogeneity of signal transduction by Na-K-ATPase α-isoforms: role of Src interaction

2018 ◽  
Vol 314 (2) ◽  
pp. C202-C210 ◽  
Author(s):  
Hui Yu ◽  
Xiaoyu Cui ◽  
Jue Zhang ◽  
Joe X. Xie ◽  
Moumita Banerjee ◽  
...  
Keyword(s):  
Binding Sites ◽  
Direct Interaction ◽  
Causative Role ◽  
Wild Type ◽  
Caveolin 1 ◽  
Proposed Model ◽  
Signaling Function ◽  
Domain Interactions ◽  
Key Residues

Of the four Na-K-ATPase α-isoforms, the ubiquitous α1 Na-K-ATPase possesses both ion transport and Src-dependent signaling functions. Mechanistically, we have identified two putative pairs of domain interactions between α1 Na-K-ATPase and Src that are critical for α1 signaling function. Our subsequent report that α2 Na-K-ATPase lacks these putative Src-binding sites and fails to carry on Src-dependent signaling further supported our proposed model of direct interaction between α1 Na-K-ATPase and Src but fell short of providing evidence for a causative role. This hypothesis was specifically tested here by introducing key residues of the two putative Src-interacting domains present on α1 but not α2 sequence into the α2 polypeptide, generating stable cell lines expressing this mutant, and comparing its signaling properties to those of α2-expressing cells. The mutant α2 was fully functional as a Na-K-ATPase. In contrast to wild-type α2, the mutant gained α1-like signaling function, capable of Src interaction and regulation. Consistently, the expression of mutant α2 redistributed Src into caveolin-1-enriched fractions and allowed ouabain to activate Src-mediated signaling cascades, unlike wild-type α2 cells. Finally, mutant α2 cells exhibited a growth phenotype similar to that of the α1 cells and proliferated much faster than wild-type α2 cells. These findings reveal the structural requirements for the Na-K-ATPase to function as a Src-dependent receptor and provide strong evidence of isoform-specific Src interaction involving the identified key amino acids. The sequences surrounding the putative Src-binding sites in α2 are highly conserved across species, suggesting that the lack of Src binding may play a physiologically important and isoform-specific role.

scholarly journals The role of Tecpr1 in selective autophagy as a cargo receptor

Autophagy ◽  
2011 ◽  
Vol 7 (11) ◽  
pp. 1389-1391 ◽  
Author(s):  
Michinaga Ogawa ◽  
Chihiro Sasakawa
Keyword(s):  
Selective Autophagy ◽  
Cargo Receptor

scholarly journals Neural Differentiation Is Inhibited through HIF1α/β-Catenin Signaling in Embryoid Bodies

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Josef Večeřa ◽  
Jana Kudová ◽  
Jan Kučera ◽  
Lukáš Kubala ◽  
Jiří Pacherník
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Direct Interaction ◽  
Embryoid Bodies ◽  
Nuclear Fraction ◽  
Wild Type ◽  
Spontaneous Differentiation

Extensive research in the field of stem cells and developmental biology has revealed evidence of the role of hypoxia as an important factor regulating self-renewal and differentiation. However, comprehensive information about the exact hypoxia-mediated regulatory mechanism of stem cell fate during early embryonic development is still missing. Using a model of embryoid bodies (EBs) derived from murine embryonic stem cells (ESC), we here tried to encrypt the role of hypoxia-inducible factor 1α (HIF1α) in neural fate during spontaneous differentiation. EBs derived from ESC with the ablated gene for HIF1α had abnormally increased neuronal characteristics during differentiation. An increased neural phenotype in Hif1α−/− EBs was accompanied by the disruption of β-catenin signaling together with the increased cytoplasmic degradation of β-catenin. The knock-in of Hif1α, as well as β-catenin ectopic overexpression in Hif1α−/− EBs, induced a reduction in neural markers to the levels observed in wild-type EBs. Interestingly, direct interaction between HIF1α and β-catenin was demonstrated by immunoprecipitation analysis of the nuclear fraction of wild-type EBs. Together, these results emphasize the regulatory role of HIF1α in β-catenin stabilization during spontaneous differentiation, which seems to be a crucial mechanism for the natural inhibition of premature neural differentiation.

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