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The American Journal of Pathology Sep 2017TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune...
TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion <30 mg/24 hours and >300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n = 9) and controls (n = 6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an in vitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During in vitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in urine, identifying the kidney as a source of sTAM.
Topics: Adult; Aged; Aged, 80 and over; Cell Line; Diabetic Nephropathies; Female; Humans; Kidney Glomerulus; Male; Middle Aged; Protein S; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; c-Mer Tyrosine Kinase; Axl Receptor Tyrosine Kinase
PubMed: 28668213
DOI: 10.1016/j.ajpath.2017.05.004 -
Philosophical Transactions of the Royal... Jun 2021Protein aggregation, particularly in its prion-like form, has long been thought to be detrimental. However, recent studies have identified multiple instances where...
Protein aggregation, particularly in its prion-like form, has long been thought to be detrimental. However, recent studies have identified multiple instances where protein aggregation is important for normal physiological functions. Combining mass spectrometry and cell biological approaches, we developed a strategy for the identification of protein aggregates in cell lysates. We used this approach to characterize prion-based traits in pathogenic strains of the yeast isolated from immunocompromised human patients. The proteins that we found, including the metabolic enzyme Cdc19, the translation elongation factor Yef3 and the fibrillarin homologue Nop1, are known to assemble under certain physiological conditions. Yet, such assemblies have not been reported to be stable or heritable. Our data suggest that some proteins which aggregate in response to stress have the capacity to acquire diverse assembled states, certain ones of which can be propagated across generations in a form of protein-based epigenetics. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'
Topics: Cell Cycle Proteins; Evolution, Molecular; Nuclear Proteins; Peptide Elongation Factors; Prions; Protein Aggregates; Pyruvate Kinase; Ribonucleoproteins, Small Nucleolar; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Physiological
PubMed: 33866806
DOI: 10.1098/rstb.2020.0127 -
The Journal of Cell Biology Mar 2019Target of rapamycin complex-2 (TORC2), a conserved protein kinase complex, is an indispensable regulator of plasma membrane homeostasis. In budding yeast (), the...
Target of rapamycin complex-2 (TORC2), a conserved protein kinase complex, is an indispensable regulator of plasma membrane homeostasis. In budding yeast (), the essential downstream effector of TORC2 is protein kinase Ypk1 and its paralog Ypk2. Muk1, a Rab5-specific guanine nucleotide exchange factor (GEF), was identified in our prior global screen for candidate Ypk1 targets. We confirm here that Muk1 is a substrate of Ypk1 and demonstrate that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Strikingly, yeast lacking its two Rab5 GEFs (Muk1 and Vps9) or its three Rab5 paralogs (Vps21/Ypt51, Ypt52, and Ypt53) or overexpressing Msb3, a Rab5-directed GTPase-activating protein, all exhibit pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Vps21 coimmunoprecipitates with TORC2, and immuno-enriched TORC2 is less active in vitro in the absence of Rab5 GTPases. Thus, TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling.
Topics: Glycogen Synthase Kinase 3; Guanine Nucleotide Exchange Factors; Mechanistic Target of Rapamycin Complex 2; Protein Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins
PubMed: 30578283
DOI: 10.1083/jcb.201807154 -
Nature Communications Jan 2017The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid...
The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large 'hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, using recombinantly purified Scc2-Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2-Scc4 uses its modular structure to make multiple contacts with cohesin.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Conserved Sequence; Models, Molecular; Protein Binding; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cohesins
PubMed: 28059076
DOI: 10.1038/ncomms13952 -
Journal of Thrombosis and Haemostasis :... Dec 2019Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and...
BACKGROUND
Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive.
OBJECTIVES
To elucidate the nature of the phospholipid-dependent interactions among APC, protein S, and FVa.
METHODS
We evaluated binding of active site blocked APC to phospholipid-coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally.
RESULTS
Activated protein C alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding (>14-fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVa (W1920R), an APC-resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex.
CONCLUSIONS
FVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions among FVa, APC, and protein S are essential for its cofactor function.
Topics: Binding Sites; Blood Coagulation; Calcium-Binding Proteins; Enzyme Activation; Factor Va; HEK293 Cells; Humans; Models, Molecular; Multiprotein Complexes; Phospholipids; Protein Binding; Protein C; Protein Conformation; Protein S; Structure-Activity Relationship; Thrombin; Thromboplastin
PubMed: 31364267
DOI: 10.1111/jth.14594 -
Scientific Reports Mar 2017Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1...
Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.
Topics: Acetylation; Acetyltransferases; Amino Acid Sequence; Animals; Binding Sites; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Crystallography, X-Ray; Gene Expression; Models, Molecular; Nuclear Proteins; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Xenopus Proteins; Xenopus laevis
PubMed: 28290497
DOI: 10.1038/srep44313 -
PLoS Genetics Aug 2017Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM...
Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM and ATR, respectively. The Tel2-Tti1-Tti2 (TTT) complex connects to the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex for the protein stability of PIKKs; however, TTT-R2TP interaction only partially mediates ATM and ATR protein stabilization. How TTT controls protein stability of ATM and ATR remains to be precisely determined. Here we show that Asa1, like Tel2, plays a major role in stabilization of newly synthesized Mec1 and Tel1 proteins whereas Pih1 contributes to Mec1 and Tel1 stability at high temperatures. Although Asa1 and Pih1 both interact with Tel2, no Asa1-Pih1 interaction is detected. Pih1 is distributed in both the cytoplasm and nucleus wheres Asa1 localizes largely in the cytoplasm. Asa1 and Pih1 are required for proper DNA damage checkpoint signaling. Our findings provide a model in which two different Tel2 pathways promote protein stabilization of Mec1 and Tel1 in budding yeast.
Topics: Ataxia Telangiectasia Mutated Proteins; DNA Damage; Intracellular Signaling Peptides and Proteins; Multiprotein Complexes; Nuclear Proteins; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Protein Stability; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Telomere-Binding Proteins
PubMed: 28827813
DOI: 10.1371/journal.pgen.1006873 -
Journal of Biological Inorganic... Sep 2019The Saccharomyces cerevisiae transcriptional activator Aft1 and its paralog Aft2 respond to iron deficiency by upregulating expression of proteins required for iron...
The Saccharomyces cerevisiae transcriptional activator Aft1 and its paralog Aft2 respond to iron deficiency by upregulating expression of proteins required for iron uptake at the plasma membrane, vacuolar iron transport, and mitochondrial iron metabolism, with the net result of mobilizing iron from extracellular sources and intracellular stores. Conversely, when iron levels are sufficient, Aft1 and Aft2 interact with the cytosolic glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, which promote Aft1/2 dissociation from DNA and subsequent export from the nucleus. Previous studies unveiled the molecular mechanism for iron-dependent inhibition of Aft1/2 activity, demonstrating that the [2Fe-2S]-bridged Grx3-Bol2 heterodimer transfers a cluster to Aft2, driving Aft2 dimerization and dissociation from DNA. Here, we provide further insight into the regulation mechanism by investigating the roles of conserved cysteines in Aft2 in iron-sulfur cluster binding and interaction with [2Fe-2S]-Grx3-Bol2. Using size exclusion chromatography and circular dichroism spectroscopy, these studies reveal that both cysteines in the conserved Aft2 Cys-Asp-Cys motif are essential for Aft2 dimerization via [2Fe-2S] cluster binding, while only one cysteine is required for interaction with the [2Fe-2S]-Grx3-Bol2 complex. Taken together, these results provide novel insight into the molecular details of iron-sulfur cluster transfer from Grx3-Bol2 to Aft2 which likely occurs through a ligand exchange mechanism. Loss of either cysteine in the Aft2 iron-sulfur binding site may disrupt this ligand-exchange process leading to the isolation of a trapped Aft2-Grx3-Bol2 intermediate, while the replacement of both cysteines abrogates both the iron-sulfur cluster exchange and the protein-protein interactions between Aft2 and Grx3-Bol2.
Topics: Chromatography, Gel; Circular Dichroism; Iron-Sulfur Proteins; Mitochondrial Proteins; Oxidoreductases; Plasmids; Protein Binding; Protein Multimerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Trans-Activators
PubMed: 31493153
DOI: 10.1007/s00775-019-01705-x -
Journal of Virology May 2022As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young...
As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.
Topics: Enterovirus A, Human; Enterovirus Infections; Glucuronosyltransferase; HeLa Cells; Heparitin Sulfate; Humans; Lysosomal Membrane Proteins; Receptors, Scavenger; Sulfate Transporters; Sulfotransferases; Tyrosine
PubMed: 35420441
DOI: 10.1128/jvi.02042-21 -
International Journal of Molecular... Nov 2019Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein...
Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in . Rpl43 protein levels are under-accumulated in the absence of or . However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in Δ and Δ are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in Δ and significantly in Δ. We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.
Topics: Binding Sites; Gene Expression Regulation, Fungal; Karyopherins; Models, Molecular; Multiprotein Complexes; Mutation; Nuclear Proteins; Protein Binding; Protein Conformation; Protein Stability; RNA-Binding Proteins; Ribosomal Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31779129
DOI: 10.3390/ijms20235941