scholarly journals Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

2020 ◽  
Author(s):  
Jiahui Tao ◽  
Amandine Berthet ◽  
Rose Citron ◽  
Robert Stanley ◽  
Jason Gestwicki ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Alpha Synuclein ◽  
Early Event ◽  
Parkinsons Disease ◽  
Interaction Site ◽  
Over Expression ◽  
Oligomer Formation ◽  
Hsp70 Chaperone ◽  
Binding Cleft ◽  
Chaperone Hsp70

Over-expression and aggregation of alpha-synuclein (ASyn) are linked to the onset and pathology of Parkinsons disease and related synucleinopathies. Elevated levels of the stress induced chaperone, Hsp70, protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirmed that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, a biological role for a non-canonical interaction was observed in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70 canonical actions. Additionally, these results raise the question of whether other misfolded proteins might also engage via the same non-canonical mechanism.

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

2021 ◽  
Vol 296 ◽  
pp. 100613
Author(s):  
Jiahui Tao ◽  
Amandine Berthet ◽  
Y. Rose Citron ◽  
Paraskevi L. Tsiolaki ◽  
Robert Stanley ◽  
...  
Keyword(s):  
Oligomer Formation ◽  
Hsp70 Chaperone

Membrane Interactions of Oligomeric Alpha-Synuclein: Potential Role in Parkinsons Disease

2010 ◽  
Vol 11 (5) ◽  
pp. 334-342 ◽  
Author(s):  
Bart D. van Rooijen ◽  
Mireille M.A.E. Claessens ◽  
Vinod Subramaniam
Keyword(s):  
Potential Role ◽  
Alpha Synuclein ◽  
Membrane Interactions ◽  
Parkinsons Disease

scholarly journals The activation loop and substrate-binding cleft of glutaminase C are allosterically coupled

2019 ◽  
Vol 295 (5) ◽  
pp. 1328-1337
Author(s):  
Yunxing Li ◽  
Sekar Ramachandran ◽  
Thuy-Tien T. Nguyen ◽  
Clint A. Stalnecker ◽  
Richard A. Cerione ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Fluorescence Emission ◽  
Reciprocal Relationship ◽  
Tryptophan Residue ◽  
Substrate Affinity ◽  
Activation Loop ◽  
Conformational Coupling ◽  
Binding Cleft ◽  
Glutaminase Activity

The glutaminase C (GAC) isoform of mitochondrial glutaminase is overexpressed in many cancer cells and therefore represents a potential therapeutic target. Understanding the regulation of GAC activity has been guided by the development of spectroscopic approaches that measure glutaminase activity in real time. Previously, we engineered a GAC protein (GAC(F327W)) in which a tryptophan residue is substituted for phenylalanine in an activation loop to explore the role of this loop in enzyme activity. We showed that the fluorescence emission of Trp-327 is enhanced in response to activator binding, but quenched by inhibitors of the BPTES class that bind to the GAC tetramer and contact the activation loop, thereby constraining it in an inactive conformation. In the present work, we took advantage of a tryptophan substitution at position 471, proximal to the GAC catalytic site, to examine the conformational coupling between the activation loop and the substrate-binding cleft, separated by ∼16 Å. Comparison of glutamine binding in the presence or absence of the BPTES analog CB-839 revealed a reciprocal relationship between the constraints imposed on the activation loop position and the affinity of GAC for substrate. Binding of the inhibitor weakened the affinity of GAC for glutamine, whereas activating anions such as Pi increased this affinity. These results indicate that the conformations of the activation loop and the substrate-binding cleft in GAC are allosterically coupled and that this coupling determines substrate affinity and enzymatic activity and explains the activities of CB-839, which is currently in clinical trials.

scholarly journals A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78

2019 ◽  
Vol 29 (14) ◽  
pp. 1689-1693 ◽  
Author(s):  
Andrew J. Ambrose ◽  
Christopher J. Zerio ◽  
Jared Sivinski ◽  
Cody J. Schmidlin ◽  
Taoda Shi ◽  
...  
Keyword(s):  
High Throughput ◽  
Binding Assay ◽  
Substrate Binding ◽  
Hsp70 Chaperone

scholarly journals Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-β-1,4-mannanase

2020 ◽  
Vol 295 (15) ◽  
pp. 5012-5021 ◽  
Author(s):  
Fernanda Mandelli ◽  
Mariana Abrahão Bueno de Morais ◽  
Evandro Antonio de Lima ◽  
Leane Oliveira ◽  
Gabriela Felix Persinoti ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Systematic Investigation ◽  
Substrate Preference ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Binding Cleft ◽  
Aromatic Cluster ◽  
Shed Light

β-Mannanases from the glycoside hydrolase 26 (GH26) family are retaining hydrolases that are active on complex heteromannans and whose genes are abundant in rumen metagenomes and metatranscriptomes. These enzymes can exhibit distinct modes of substrate recognition and are often fused to carbohydrate-binding modules (CBMs), resulting in a molecular puzzle of mechanisms governing substrate preference and mode of action that has not yet been pieced together. In this study, we recovered a novel GH26 enzyme with a CBM35 module linked to its N terminus (CrMan26) from a cattle rumen metatranscriptome. CrMan26 exhibited a preference for galactomannan as substrate and the crystal structure of the full-length protein at 1.85 Å resolution revealed a unique orientation of the ancillary domain relative to the catalytic interface, strategically positioning a surface aromatic cluster of the ancillary domain as an extension of the substrate-binding cleft, contributing to galactomannan preference. Moreover, systematic investigation of nonconserved residues in the catalytic interface unveiled that residues Tyr195 (−3 subsite) and Trp234 (−5 subsite) from distal negative subsites have a key role in galactomannan preference. These results indicate a novel and complex mechanism for substrate recognition involving spatially remote motifs, distal negative subsites from the catalytic domain, and a surface-associated aromatic cluster from the ancillary domain. These findings expand our molecular understanding of the mechanisms of substrate binding and recognition in the GH26 family and shed light on how some CBMs and their respective orientation can contribute to substrate preference.

scholarly journals Alpha-Synuclein and LRRK2 in Synaptic Autophagy: Linking Early Dysfunction to Late-Stage Pathology in Parkinson’s Disease

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1115 ◽  
Author(s):  
Giulia Lamonaca ◽  
Mattia Volta
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Late Stage ◽  
Disease Onset ◽  
Disease Process ◽  
Alpha Synuclein ◽  
Synaptic Activity ◽  
Early Event ◽  
Cellular Targets ◽  
Disease Modifying

The lack of effective disease-modifying strategies is the major unmet clinical need in Parkinson’s disease. Several experimental approaches have attempted to validate cellular targets and processes. Of these, autophagy has received considerable attention in the last 20 years due to its involvement in the clearance of pathologic protein aggregates and maintenance of neuronal homeostasis. However, this strategy mainly addresses a very late stage of the disease, when neuropathology and neurodegeneration have likely “tipped over the edge” and disease modification is extremely difficult. Very recently, autophagy has been demonstrated to modulate synaptic activity, a process distinct from its catabolic function. Abnormalities in synaptic transmission are an early event in neurodegeneration with Leucine-Rich Repeat Kinase 2 (LRRK2) and alpha-synuclein strongly implicated. In this review, we analyzed these processes separately and then discussed the unification of these biomolecular fields with the aim of reconstructing a potential “molecular timeline” of disease onset and progression. We postulate that the elucidation of these pathogenic mechanisms will form a critical basis for the design of novel, effective disease-modifying therapies that could be applied early in the disease process.

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