scholarly journals Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

2008 ◽  
Vol 19 (8) ◽  
pp. 3514-3525 ◽  
Author(s):  
Masato Takeuchi ◽  
Yukio Kimata ◽  
Kenji Kohno
Keyword(s):  
Molecular Chaperones ◽  
Secretory Pathway ◽  
Temperature Sensitive ◽  
Type I ◽  
Accelerated Degradation ◽  
Transient Interaction ◽  
Protein Functions ◽  
Substrate Proteins

Molecular chaperones prevent aggregation of denatured proteins in vitro and are thought to support folding of diverse proteins in vivo. Chaperones may have some selectivity for their substrate proteins, but knowledge of particular in vivo substrates is still poor. We here show that yeast Rot1, an essential, type-I ER membrane protein functions as a chaperone. Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels. Furthermore, we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins. Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. Their folding also requires BiP, and one of these proteins was simultaneously associated with both Rot1 and BiP, suggesting that they can cooperate to facilitate protein folding. The Rot1-dependent proteins include a soluble, type I and II, and polytopic membrane proteins, and they do not share structural similarities. In addition, their dependency on Rot1 appeared different. We therefore propose that Rot1 is a general chaperone with some substrate specificity.

scholarly journals Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
Author(s):  
Sarah L. Barker ◽  
Linda Lee ◽  
B. Daniel Pierce ◽  
Lymarie Maldonado-Báez ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

scholarly journals Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.

1991 ◽  
Vol 114 (4) ◽  
pp. 671-679 ◽  
Author(s):  
T Oka ◽  
S Nishikawa ◽  
A Nakano
Keyword(s):  
Temperature Sensitivity ◽  
Protein Function ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Transport Reaction ◽  
Golgi Transport ◽  
Gtp Binding

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3719-3729 ◽  
Author(s):  
Katrijn Bockstael ◽  
Nick Geukens ◽  
Lieve Van Mellaert ◽  
Piet Herdewijn ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Functional Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antimicrobial Treatment ◽  
Signal Peptidase ◽  
Temperature Sensitive ◽  
Type I

The development of antibacterial resistance is inevitable and is a major concern in hospitals and communities. Moreover, biofilm-grown bacteria are less sensitive to antimicrobial treatment. In this respect, the Gram-positive Staphylococcus epidermidis is an important source of nosocomial biofilm-associated infections. In the search for new antibacterial therapies, the type I signal peptidase (SPase I) serves as a potential target for development of antibacterials with a novel mode of action. This enzyme cleaves off the signal peptide from secreted proteins, making it essential for protein secretion, and hence for bacterial cell viability. S. epidermidis encodes three putative SPases I (denoted Sip1, Sip2 and Sip3), of which Sip1 lacks the catalytic lysine. In this report, we investigated the active S. epidermidis SPases I in more detail. Sip2 and Sip3 were found to complement a temperature-sensitive Escherichia coli lepB mutant, demonstrating their in vivo functional activity. In vitro functional activity of purified Sip2 and Sip3 proteins and inhibition of their activity by the SPase I inhibitor arylomycin A2 were further illustrated using a fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we demonstrated that SPase I not only is an attractive target for development of novel antibacterials against free-living bacteria, but also is a feasible target for biofilm-associated infections.

Peculiarities of the course of type I allergic reactions in patients with blood diseases

2020 ◽  
pp. 40-50
Author(s):  
A. Nikitina
Keyword(s):  
Search Engines ◽  
Anaphylactic Shock ◽  
Type I ◽  
Allergic Reactions ◽  
Common Cause ◽  
Blood Diseases ◽  
Treatment Regimens ◽  
Modern Methods

Analysis of literature data presented in search engines — Elibrary, PubMed, Cochrane — concerning the risk of developing type I allergic reactions in patients with blood diseases is presented. It is shown that the most common cause of type I allergic reactions is drugs included in the treatment regimens of this category of patients. The article presents statistics on the increase in the number of drug allergies leading to cases of anaphylactic shock in patients with blood diseases. Modern methods for the diagnosis of type I allergic reactions in vivo and in vitro are considered.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Gao ◽  
Xianwei Ma ◽  
Ming Yuan ◽  
Yulan Yi ◽  
Guoke Liu ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Type I Interferon ◽  
Zinc Fingers ◽  
E3 Ligase ◽  
Type I ◽  
E3 Ligases ◽  
Protein Posttranslational Modifications ◽  
Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

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