scholarly journals Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular ageing research

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kara L. Schneider ◽  
Adam J. M. Wollman ◽  
Thomas Nyström ◽  
Sviatlana Shashkova
Keyword(s):  
Quality Control ◽  
Experimental Evidence ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stress Protein ◽  
Protein Quality Control ◽  
Ageing Research ◽  
Fluorescent Tags ◽  
And Function

AbstractThe yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.

scholarly journals Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular aging research

2021 ◽  
Author(s):  
Kara L. Schneider ◽  
Adam Wollman ◽  
Thomas Nyström ◽  
Sviatlana Shashkova
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stress Protein ◽  
Protein Quality Control ◽  
Cellular Aging ◽  
Aging Research ◽  
Ageing Research ◽  
Microbiology Research

AbstractThe yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing endogenously expressed Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. Overall, such protein fusions used in molecular and microbiology research are often sparsely characterised. To address this issue, we performed a comparative assessment of Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on the fluorophore within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects and points may play a role in other eukaryotic systems.

Abstract 85: The AAA-ATPase p97 is a Critical Regulator of Cardiac Homeostasis

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Matthew J Brody ◽  
Michelle A Sargent ◽  
Jeffery D Molkentin
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Complexes ◽  
Protein Quality Control ◽  
Ubiquitin Proteasome System ◽  
Cellular Protein ◽  
Dominant Negative ◽  
Aaa Atpase ◽  
And Function ◽  
Thrombospondin 4

p97 is a AAA-ATPase that plays critical roles in a myriad of cellular protein quality control processes, including the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway that targets misfolded proteins in the ER for degradation in the cytosol by the ubiquitin proteasome system. Mutations in p97 cause a multisystem degenerative proteinopathy disorder called inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) that includes pathologies of the nervous system, skeletal muscle, bone, and heart. Previous studies in the laboratory into the mechanisms whereby thrombospondin 4 has its cardioprotective effects and enhanced ERAD activity identified p97 as a direct interacting partner. This observation suggested that p97 itself could be an important cardioprotective effector by benefiting protein quality control in the heart. To address this hypothesis here we generated cardiac-specific transgenic mice overexpressing wildtype p97 or a p97 K524A mutant with deficient ATPase activity, the latter of which functioned as a dominant negative. Mice overexpressing wildtype p97 exhibit normal cardiac structure and function while mutant p97 overexpressing mice develop cardiomyopathy, upregulate several ERAD complex components, and have elevated levels of ubiquitinated proteins. Proteomics and immunoprecipitation assays identified overwhelming interactions between endogenous p97 and a number of interesting protein complexes that suggest unique functions for this protein in regulating protein quality control in the heart. The results and novel regulatory relationships will be presented, which suggests entirely unique pathways whereby p97 functions in the heart.

scholarly journals Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation

2019 ◽  
Vol 20 (24) ◽  
pp. 6220 ◽  
Author(s):  
Joan Castells-Ballester ◽  
Natalie Rinis ◽  
Ilgin Kotan ◽  
Lihi Gal ◽  
Daniela Bausewein ◽  
...  
Keyword(s):  
Quality Control ◽  
Translational Regulation ◽  
Secretory Pathway ◽  
Rna Binding ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Protein Quality Control ◽  
Ribosome Profiling ◽  
Unfolded Protein

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

323-OR: Synergism between Protein Quality Control Pathways to Maintain Alpha-Cell Mass and Function

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 323-OR
Author(s):  
RACHEL B. REINERT ◽  
XIANWEI CUI ◽  
NEHA SHRESTHA ◽  
LING QI
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Cell Mass ◽  
Protein Quality Control ◽  
Alpha Cell ◽  
And Function

scholarly journals Neddylation, an Emerging Mechanism Regulating Cardiac Development and Function

2020 ◽  
Vol 11 ◽  
Author(s):  
Jie Li ◽  
Jianqiu Zou ◽  
Rodney Littlejohn ◽  
Jinbao Liu ◽  
Huabo Su
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Cardiac Development ◽  
Current Knowledge ◽  
Biological Significance ◽  
Protein Quality Control ◽  
Pathogenic Factors ◽  
Health And Disease ◽  
The Stability ◽  
And Function

Defects in protein quality control have been increasingly recognized as pathogenic factors in the development of heart failure, a persistent devastating disease lacking efficacious therapies. Ubiquitin and ubiquitin-like proteins, a family of post-translational modifying polypeptides, play important roles in controlling protein quality by maintaining the stability and functional diversity of the proteome. NEDD8 (neural precursor cell expressed, developmentally downregulated 8), a small ubiquitin-like protein, was discovered two decades ago but until recently the biological significance of NEDD8 modifications (neddylation) in the heart has not been appreciated. In this review, we summarize the current knowledge of the biology of neddylation, highlighting several mechanisms by which neddylation regulates the function of its downstream targets, and discuss the expanding roles for neddylation in cardiac physiology and disease, with an emphasis on cardiac protein quality control. Finally, we outline challenges linked to the study of neddylation in health and disease.

Protein quality control at the endoplasmic reticulum

2016 ◽  
Vol 60 (2) ◽  
pp. 227-235 ◽  
Author(s):  
Kathleen McCaffrey ◽  
Ineke Braakman
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Quality ◽  
Bond Formation ◽  
Protein Quality Control ◽  
Secretory Proteins ◽  
Protein Quality Control System ◽  
And Function ◽  
Extracellular Environment

The ER (endoplasmic reticulum) is the protein folding ‘factory’ of the secretory pathway. Virtually all proteins destined for the plasma membrane, the extracellular space or other secretory compartments undergo folding and maturation within the ER. The ER hosts a unique PQC (protein quality control) system that allows specialized modifications such as glycosylation and disulfide bond formation essential for the correct folding and function of many secretory proteins. It is also the major checkpoint for misfolded or aggregation-prone proteins that may be toxic to the cell or extracellular environment. A failure of this system, due to aging or other factors, has therefore been implicated in a number of serious human diseases. In this article, we discuss several key features of ER PQC that maintain the health of the cellular secretome.

scholarly journals Methionine Redox Homeostasis in Protein Quality Control

2021 ◽  
Vol 8 ◽  
Author(s):  
Laurent Aussel ◽  
Benjamin Ezraty
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Protein Function ◽  
Protein Quality ◽  
Redox Homeostasis ◽  
Protein Quality Control ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductases ◽  
Methionine Residues ◽  
And Function

Bacteria live in different environments and are subject to a wide variety of fluctuating conditions. During evolution, they acquired sophisticated systems dedicated to maintaining protein structure and function, especially during oxidative stress. Under such conditions, methionine residues are converted into methionine sulfoxide (Met-O) which can alter protein function. In this review, we focus on the role in protein quality control of methionine sulfoxide reductases (Msr) which repair oxidatively protein-bound Met-O. We discuss our current understanding of the importance of Msr systems in rescuing protein function under oxidative stress and their ability to work in coordination with chaperone networks. Moreover, we highlight that bacterial chaperones, like GroEL or SurA, are also targeted by oxidative stress and under the surveillance of Msr. Therefore, integration of methionine redox homeostasis in protein quality control during oxidative stress gives a complete picture of this bacterial adaptive mechanism.

scholarly journals Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery

2020 ◽  
Vol 477 (18) ◽  
pp. 3471-3497 ◽  
Author(s):  
Tongyin Zheng ◽  
Yiran Yang ◽  
Carlos A. Castañeda
Keyword(s):  
Quality Control ◽  
Rna Binding ◽  
Protein Quality ◽  
Rna Binding Proteins ◽  
Protein Quality Control ◽  
Ubiquitin Proteasome System ◽  
Protein Homeostasis ◽  
Intrinsically Disordered ◽  
Structure Dynamics ◽  
And Function

Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin–proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.

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