scholarly journals PES inhibits human inducible Hsp70 by covalent targeting of cysteine residues in the substrate binding domain

2020 ◽  
pp. jbc.RA120.015440
Author(s):  
Jie Yang ◽  
Weibin Gong ◽  
Si Wu ◽  
Hong Zhang ◽  
Sarah Perrett
Keyword(s):  
Drug Target ◽  
Protein Quality ◽  
Addition Reaction ◽  
Conformational Dynamics ◽  
Covalent Attachment ◽  
Michael Addition Reaction ◽  
Cellular Processes ◽  
Covalent Inhibitors ◽  
And Function ◽  
Substrate Binding Domain

Hsp70 proteins are a family of ancient and conserved chaperones. They play important roles in vital cellular processes, such as protein quality control and the stress response. Hsp70 proteins are a potential drug target for treatment of disease, particularly cancer. PES (2-phenylethynesulfonamide or pifithrin-μ) has been reported to be an inhibitor of Hsp70. However, the mechanism of PES inhibition is still unclear. In this study we found that PES can undergo a Michael addition reaction with Cys-574 and Cys-603 in the SBDα of hHsp70, resulting in covalent attachment of a PES molecule to each Cys residue. We previously showed that glutathionylation of Cys-574 and Cys-603 affects the structure and function of hHsp70. In this study, PES modification showed similar structural and functional effects on hHsp70 to glutathionylation. Further, we found that susceptibility to PES modification is influenced by changes in the conformational dynamics of the SBDα, such as are induced by interaction with adjacent domains, allosteric changes and mutations. This study provides new avenues for development of covalent inhibitors of hHsp70.

scholarly journals Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

2020 ◽  
Author(s):  
Matthias Fellner ◽  
Rinky Parakra ◽  
Kirstin O. McDonald ◽  
Itamar Kass ◽  
Guy N.L. Jameson ◽  
...  
Keyword(s):  
Peroxidase Activity ◽  
Conformational Dynamics ◽  
Cellular Processes ◽  
Pathogenic Variants ◽  
Alkaline Transition ◽  
Apoptotic Activity ◽  
Dynamics Simulations ◽  
And Function ◽  
Platelet Formation

AbstractMutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with exchange between conformational states proposed to regulate changes in function. Here we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. We find that three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with increased overall flexibility of cytochrome c, particularly movement of the Ω loops. This suggests that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an “induced fit” mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics suggesting that the mechanism by which the variants alter peroxidase activity is not related to the conformation dynamics of the hexacoordinate heme Fe state of cytochrome c analyzed in the simulations. Recent suggestions that conformational mobility of specific regions of cytochrome c underpins changes in reduction potential and the alkaline transition pK were not supported. These data highlight that conformational dynamics of cytochrome c drives some but not all of its properties and activities.

scholarly journals The structure and function of deubiquitinases: lessons from budding yeast

Open Biology ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 200279
Author(s):  
Harsha Garadi Suresh ◽  
Natasha Pascoe ◽  
Brenda Andrews
Keyword(s):  
Protein Quality ◽  
Budding Yeast ◽  
Post Translational Modification ◽  
Repair Gene ◽  
Cellular Processes ◽  
Protein Ubiquitination ◽  
Damage Repair ◽  
The Many ◽  
Key Aspects ◽  
And Function

Protein ubiquitination is a key post-translational modification that regulates diverse cellular processes in eukaryotic cells. The specificity of ubiquitin (Ub) signalling for different bioprocesses and pathways is dictated by the large variety of mono-ubiquitination and polyubiquitination events, including many possible chain architectures. Deubiquitinases (DUBs) reverse or edit Ub signals with high sophistication and specificity, forming an integral arm of the Ub signalling machinery, thus impinging on fundamental cellular processes including DNA damage repair, gene expression, protein quality control and organellar integrity. In this review, we discuss the many layers of DUB function and regulation, with a focus on insights gained from budding yeast. Our review provides a framework to understand key aspects of DUB biology.

scholarly journals Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones

2020 ◽  
Vol 295 (21) ◽  
pp. 7301-7316 ◽  
Author(s):  
Despina Serlidaki ◽  
Maria A. W. H. van Waarde ◽  
Lukas Rohland ◽  
Anne S. Wentink ◽  
Suzanne L. Dekker ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Protein Quality ◽  
Nucleotide Exchange ◽  
Protein Variant ◽  
Cellular Processes ◽  
Nucleotide Exchange Factor ◽  
Opposing Effects ◽  
Substrate Binding Domain ◽  
Lateral Sclerosis

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis–associated protein variant superoxide dismutase 1 (SOD1)–A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2.

The Complexity of DEK Signaling in Cancer Progression

2018 ◽  
Vol 18 (3) ◽  
pp. 256-265 ◽  
Author(s):  
Yong Teng ◽  
Liwei lang ◽  
Catherine E. Jauregui
Keyword(s):  
Cancer Progression ◽  
Drug Target ◽  
Diagnostic Marker ◽  
Cell Phenotype ◽  
Regulation Of Transcription ◽  
Cellular Processes ◽  
Human Genes ◽  
Cancer Initiation ◽  
Chromatin Structural ◽  
And Function

The DNA binding protein and chromatin structural regulator DEK regulate many cellular processes. These include proliferation, differentiation, apoptosis, senescence, DNA repairing and the maintenance of stem cell phenotype. DEK is increasingly recognized as a crucial player in many steps of cancer initiation and progression, and is precisely regulated by abundant promoting and inhibiting factors directly or indirectly. DEK may serve as an architectural modulating protein to regulate the expression and function of multiple human genes in cancer cells. In this article we have reviewed the specificities and complexities of DEK in the regulation of transcription factors and global chromatin, including its biologic roles in malignant cells, and summarized the current research. The possible use of DEK as a diagnostic marker and drug target in the prevention or treatment of tumors is also discussed.

SUMO and ubiquitin paths converge

2010 ◽  
Vol 38 (1) ◽  
pp. 34-39 ◽  
Author(s):  
Amanda Denuc ◽  
Gemma Marfany
Keyword(s):  
Genome Stability ◽  
Cell Signalling ◽  
Covalent Attachment ◽  
Small Peptides ◽  
Protein Activity ◽  
Protein Protein Interaction ◽  
Cellular Processes ◽  
Reversible Covalent ◽  
And Function

One of the more rapidly expanding fields in cell signalling nowadays is the characterization of proteins conjugated to Ub (ubiquitin) or Ub-like peptides, such as SUMO (small Ub-related modifier). The reversible covalent attachment of these small peptides remodels the target protein, providing new protein–protein interaction interfaces, which can be dynamically regulated given a set of enzymes for conjugation and deconjugation. First, ubiquitination was thought to be merely relegated to the control of protein turnover and degradation, whereas the attachment of SUMO was involved in the regulation of protein activity and function. However, the boundaries between the protein fates related to these tag molecules are becoming more and more fuzzy, as either the differences between mono-, multi- and poly-modifications or the lysine residue used for growth of the poly-chains is being dissected. The Ub and SUMO pathways are no longer separated, and many examples of this cross-talk are found in the literature, involving different cellular processes ranging from DNA repair and genome stability, to the regulation of protein subcellular localization or enzyme activity. Here, we review several cases in which SUMOylation and ubiquitination intersect, showing also that the same protein can be conjugated to SUMO and Ub for antagonistic, synergistic or multiple outcomes, illustrating the intricacy of the cellular signalling networks. Ub and SUMO have met and are now applying for new regulatory roles in the cell.

scholarly journals Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

2021 ◽  
Vol 478 (3) ◽  
pp. 669-684
Author(s):  
Matthias Fellner ◽  
Rinky Parakra ◽  
Kirstin O. McDonald ◽  
Itamar Kass ◽  
Guy N.L. Jameson ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Cytochrome C ◽  
Peroxidase Activity ◽  
Conformational Dynamics ◽  
Cellular Processes ◽  
Pathogenic Variants ◽  
Alkaline Transition ◽  
Apoptotic Activity ◽  
Dynamics Simulations ◽  
And Function

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an ‘induced fit’ mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

scholarly journals Bend, Push, Stretch: Remarkable Structure and Mechanics of Single Intermediate Filaments and Meshworks

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1960
Author(s):  
K. Tanuj Sapra ◽  
Ohad Medalia
Keyword(s):  
Intermediate Filaments ◽  
Eukaryotic Cell ◽  
Coiled Coils ◽  
Cellular Functions ◽  
Mechanical Pressure ◽  
Mechanical Feature ◽  
Cellular Processes ◽  
Nuclear Lamins ◽  
Filament Networks ◽  
And Function

The cytoskeleton of the eukaryotic cell provides a structural and functional scaffold enabling biochemical and cellular functions. While actin and microtubules form the main framework of the cell, intermediate filament networks provide unique mechanical properties that increase the resilience of both the cytoplasm and the nucleus, thereby maintaining cellular function while under mechanical pressure. Intermediate filaments (IFs) are imperative to a plethora of regulatory and signaling functions in mechanotransduction. Mutations in all types of IF proteins are known to affect the architectural integrity and function of cellular processes, leading to debilitating diseases. The basic building block of all IFs are elongated α-helical coiled-coils that assemble hierarchically into complex meshworks. A remarkable mechanical feature of IFs is the capability of coiled-coils to metamorphize into β-sheets under stress, making them one of the strongest and most resilient mechanical entities in nature. Here, we discuss structural and mechanical aspects of IFs with a focus on nuclear lamins and vimentin.

scholarly journals Difluorocarbene enables to access 2-fluoroindoles from ortho-vinylanilines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jianke Su ◽  
Xinyuan Hu ◽  
Hua Huang ◽  
Yu Guo ◽  
Qiuling Song
Keyword(s):  
Fluorine Atom ◽  
High Efficiency ◽  
Addition Reaction ◽  
Bond Cleavage ◽  
Therapeutic Agents ◽  
Bioactive Molecules ◽  
General Strategy ◽  
Michael Addition Reaction ◽  
Fundamental Properties ◽  
Synthetic Approaches

Abstract2-Fluoroindoles as an important structural scaffold are widely existing in many bioactive or therapeutic agents. Despite their potential usefulness, efficient constructions of 2-fluoroindole derivatives are very sparce. The development of straightforward synthetic approaches to access 2-fluoroindoles is highly desirable for studying their fundamental properties and applications. Herein, we report an efficient and general strategy for the construction of 2-fluoroindoles in which a wide variety of 2-fluoroindoles were accessed with high efficiency and chemoselectivity. Instead of starting from indole skeletons, our strategy constructs indole scaffolds alongside the incorporation of fluorine atom on C2 position in a formal [4+1] cyclization from readily accessible ortho-vinylanilines and difluorocarbene. In our protocol, commercially accessible halodifluoroalkylative reagents provide one carbon and one fluorine atom by cleaving one C-N tertiary bond and forming one C-N bond and one C-C double bond with ortho-vinylanilines. Downstream transformations on 2-fluoroindoles lead to various valuable bioactive molecules which demonstrated significant synthetic advantages over previous reports. And mechanistic studies suggest that the reaction undergoes a cascade difluorocarbene-trapping and intramolecular Michael addition reaction followed by Csp3-F bond cleavage.

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