scholarly journals Collagen I weakly interacts with the β-sheets of β2-microglobulin and enhances conformational exchange to induce amyloid formation

2019 ◽  
Author(s):  
Cody L. Hoop ◽  
Jie Zhu ◽  
Shibani Bhattacharya ◽  
Caitlyn A. Tobita ◽  
Sheena E. Radford ◽  
...  
Keyword(s):  
Protein Function ◽  
Weak Interactions ◽  
Nmr Relaxation ◽  
Conformational Exchange ◽  
Collagen I ◽  
Solution Nmr ◽  
Amyloid Formation ◽  
Relaxation Methods ◽  
A Minor

ABSTRACTAmyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial unfolding. However, how this unfolding event is triggered for proteins that are otherwise stable in their native environments is not well understood. The accumulation of the immunoglobulin protein β2-microglobulin (β2m) into amyloid plaques in the joints of long-term hemodialysis patients is the hallmark of Dialysis Related Amyloidosis (DRA). While β2m does not form amyloid unassisted near neutral pH in vitro, the localization of β2m deposits to joint spaces suggests a role for the local extracellular matrix (ECM) proteins, specifically collagens, in promoting amyloid formation. Indeed, collagen and other ECM components have been observed to facilitate β2m amyloid formation, but the large size and anisotropy of the complex, combined with the low affinity of these interactions, has limited atomic-level elucidation of the amyloid-promoting mechanism by these molecules. Using solution NMR approaches that uniquely probe weak interactions and large complexes, we are able to derive binding interfaces for collagen I on β2m and detect collagen I-induced µs–ms timescale dynamics in the β2m backbone. By combining solution NMR relaxation methods and 15N-dark state exchange saturation transfer experiments, we propose a model in which weak, multimodal collagen I–β2m interactions promote exchange with a minor population of an amyloid-competent species to induce fibrillogenesis. The results portray the intimate role of the environment in switching an innocuous protein into an amyloid-competent state, rationalizing the localization of amyloid deposits in DRA.

scholarly journals Collagen I Weakly Interacts with the β-Sheets of β2-Microglobulin and Enhances Conformational Exchange To Induce Amyloid Formation

2019 ◽  
Vol 142 (3) ◽  
pp. 1321-1331
Author(s):  
Cody L. Hoop ◽  
Jie Zhu ◽  
Shibani Bhattacharya ◽  
Caitlyn A. Tobita ◽  
Sheena E. Radford ◽  
...  
Keyword(s):  
Conformational Exchange ◽  
Collagen I ◽  
Amyloid Formation ◽  
Β2 Microglobulin ◽  
Β Sheets

scholarly journals Structural origins of protein conformational entropy

2021 ◽  
Author(s):  
José A. Caro ◽  
Kathleen G. Valentine ◽  
A. Joshua Wand
Keyword(s):  
Ligand Binding ◽  
Protein Function ◽  
Nmr Relaxation ◽  
Internal Motion ◽  
Side Chains ◽  
Conformational Entropy ◽  
Relaxation Methods ◽  
Detailed Structural Analysis ◽  
Packing Defects ◽  
Single Water Molecule

AbstractThe thermodynamics of molecular recognition by proteins is a central determinant of complex biochemistry. For over a half-century detailed cryogenic structures have provided deep insight into the energetic contributions to ligand binding by proteins1. More recently, a dynamical proxy based on NMR-relaxation methods has revealed an unexpected richness in the contributions of conformational entropy to the thermodynamics of ligand binding2,3,4,5. There remains, however, a discomforting absence of an understanding of the structural origins of fast internal motion and the conformational entropy that this motion represents. Here we report the pressure-dependence of fast internal motion within the ribonuclease barnase and its complex with the protein barstar. Distinctive clustering of the pressure sensitivity correlates with the presence of small packing defects or voids surrounding affected side chains. Prompted by this observation, we performed an analysis of the voids surrounding over 2,500 methyl-bearing side chains having experimentally determined order parameters. We find that changes in unoccupied volume as small as a single water molecule surrounding buried side chains greatly affects motion on the subnanosecond timescale. The discovered relationship begins to permit construction of a united view of the relationship between changes in the internal energy, as exposed by detailed structural analysis, and the conformational entropy, as represented by fast internal motion, in the thermodynamics of protein function.

scholarly journals Mapping transiently formed and sparsely populated conformations on a complex energy landscape

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Yong Wang ◽  
Elena Papaleo ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Energy Landscape ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Conformational Exchange ◽  
Internal Cavity ◽  
T4 Lysozyme ◽  
Enhanced Sampling ◽  
Exchange Processes ◽  
Dynamics Simulations ◽  
A Minor

Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally.

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.

Elucidating the anti-biofilm and anti-quorum sensing potential of selenocystine against respiratory tract infections causing bacteria: in vitro and in silico studies

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Bharti Patel ◽  
Subrata Mishra ◽  
Indira K. Priyadarsini ◽  
Sirisha L. Vavilala
Keyword(s):  
Quorum Sensing ◽  
Respiratory Tract ◽  
Protein Function ◽  
Docking Studies ◽  
Klebsiella Pneumonia ◽  
Potential Candidate ◽  
In Silico Studies ◽  
Specific Proteins ◽  
Tract Infections

Abstract Bacteria are increasingly relying on biofilms to develop resistance to antibiotics thereby resulting in their failure in treating many infections. In spite of continuous research on many synthetic and natural compounds, ideal anti-biofilm molecule is still not found thereby warranting search for new class of molecules. The current study focuses on exploring anti-biofilm potential of selenocystine against respiratory tract infection (RTI)-causing bacteria. Anti-bacterial and anti-biofilm assays demonstrated that selenocystine inhibits the growth of bacteria in their planktonic state, and formation of biofilms while eradicating preformed-biofilm effectively. Selenocystine at a MIC50 as low as 42 and 28 μg/mL effectively inhibited the growth of Klebsiella pneumonia and Pseudomonas aeruginosa. The antibacterial effect is further reconfirmed by agar cup diffusion assay and growth-kill assay. Selenocystine showed 30–60% inhibition of biofilm formation in K. pneumonia, and 44–70% in P. aeruginosa respectively. It also distorted the preformed-biofilms by degrading the eDNA component of the Extracellular Polymeric Substance matrix. Molecular docking studies of selenocystine with quorum sensing specific proteins clearly showed that through the carboxylic acid moiety it interacts and inhibits the protein function, thereby confirming its anti-biofilm potential. With further validation selenocystine can be explored as a potential candidate for the treatment of RTIs.

scholarly journals In-Vitro Evaluation of the Antioxidant and Anti-Inflammatory Activity of Volatile Compounds and Minerals in Five Different Onion Varieties

Separations ◽  
2021 ◽  
Vol 8 (5) ◽  
pp. 57
Author(s):  
Rokayya Sami ◽  
Abeer Elhakem ◽  
Mona Alharbi ◽  
Manal Almatrafi ◽  
Nada Benajiba ◽  
...  
Keyword(s):  
Volatile Compounds ◽  
Dimethyl Sulfide ◽  
Antioxidant Activities ◽  
Dry Weight ◽  
No Production ◽  
Oxidative Stresses ◽  
Anti Inflammatory ◽  
Red Variety ◽  
A Minor

Onions contain high antioxidants compounds that fight inflammation against many diseases. The purpose was to investigate some selected bioactive activities of onion varieties (Yellow, Red, Green, Leek, and Baby). Antioxidant assays and anti-inflammatory activities such as NO production with the addition of some bioactive components were determined and analyzed by using a spectrophotometer. Gas chromatography and mass spectrometry (GC–MS) was used for the volatile compounds, while an Atomic absorption spectrometer was used for mineral determinations. Red variety achieved the highest antioxidant activities. The total flavonoids were between (12.56 and 353.53 mg Quercetin/gin dry weight) (dw) and the total phenol was (8.75–25.73 mg/g dw). Leek, Yellow and Green extracts achieved highly anti-inflammatory values (3.71–4.01 μg/mL) followed by Red and Baby extracts, respectively. The highest contents of sodium, potassium, zinc, and calcium were established for Red onions. Furfuraldehyde, 5-Methyl-2-furfuraldehyde, 2-Methyl-2-pentenal, and 1-Propanethiol were the most predominant, followed by a minor abundance of the other compounds such as Dimethyl sulfide, Methyl allyl disulfide, Methyl-trans-propenyl-disulfide, and Methyl propyl disulfide. The results recommend that these varieties could act as sources of essential antioxidants and anti-inflammatories to decrease inflammation and oxidative stresses, especially red onions that recorded high activities.

scholarly journals A study of deregulated MMR pathways and anticancer potential of curcuma derivatives using computational approach

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Priyanjali Bhattacharya ◽  
Trupti N. Patel
Keyword(s):  
Mismatch Repair ◽  
Protein Function ◽  
Curcuma Longa ◽  
Signaling Cascades ◽  
Altered Expression ◽  
Anticancer Properties ◽  
Medicinal Properties ◽  
Multiple Signaling

AbstractPlant derived products have steadily gained momentum in treatment of cancer over the past decades. Curcuma and its derivatives, in particular, have diverse medicinal properties including anticancer potential with proven safety as supported by numerous in vivo and in vitro studies. A defective Mis-Match Repair (MMR) is implicated in solid tumors but its role in haematologic malignancies is not keenly studied and the current literature suggests that it is limited. Nonetheless, there are multiple pathways interjecting the mismatch repair proteins in haematologic cancers that may have a direct or indirect implication in progression of the disease. Here, through computational analysis, we target proteins that are involved in rewiring of multiple signaling cascades via altered expression in cancer using various curcuma derivatives (Curcuma longa L. and Curcuma caesia Roxb.) which in turn, profoundly controls MMR protein function. These biomolecules were screened to identify their efficacy on selected targets (in blood-related cancers); aberrations of which adversely impacted mismatch repair machinery. The study revealed that of the 536 compounds screened, six of them may have the potential to regulate the expression of identified targets and thus revive the MMR function preventing genomic instability. These results reveal that there may be potential plant derived biomolecules that may have anticancer properties against the tumors driven by deregulated MMR-pathways.

scholarly journals Inhibition of Lysine 63 Ubiquitination Prevents the Progression of Renal Fibrosis in Diabetic DBA/2J Mice

2021 ◽  
Vol 22 (10) ◽  
pp. 5194
Author(s):  
Paola Pontrelli ◽  
Francesca Conserva ◽  
Rossella Menghini ◽  
Michele Rossini ◽  
Alessandra Stasi ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Epithelial To Mesenchymal Transition ◽  
Selective Inhibition ◽  
Collagen I ◽  
Protein Accumulation ◽  
Mesenchymal Transition ◽  
Collagen Iii ◽  
Proximal Tubular Epithelial Cells

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

scholarly journals Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism: novel aspects

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Benedikt Frieg ◽  
Boris Görg ◽  
Holger Gohlke ◽  
Dieter Häussinger
Keyword(s):  
Glutamine Synthetase ◽  
Protein Function ◽  
Nitrosative Stress ◽  
Point Mutations ◽  
Central Element ◽  
Ph Sensitive ◽  
Tyrosine Nitration ◽  
Ammonia Metabolism ◽  
Functional Consequences

Abstract Glutamine synthetase (GS) in the liver is expressed in a small perivenous, highly specialized hepatocyte population and is essential for the maintenance of low, non-toxic ammonia levels in the organism. However, GS activity can be impaired by tyrosine nitration of the enzyme in response to oxidative/nitrosative stress in a pH-sensitive way. The underlying molecular mechanism as investigated by combined molecular simulations and in vitro experiments indicates that tyrosine nitration can lead to a fully reversible and pH-sensitive regulation of protein function. This approach was also used to understand the functional consequences of several recently described point mutations of human GS with clinical relevance and to suggest an approach to restore impaired GS activity.

scholarly journals Novel FGFR1 Variants Are Associated with Congenital Scoliosis

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1126
Author(s):  
Shengru Wang ◽  
Xiran Chai ◽  
Zihui Yan ◽  
Sen Zhao ◽  
Yang Yang ◽  
...  
Keyword(s):  
Protein Function ◽  
Hypogonadotropic Hypogonadism ◽  
Congenital Scoliosis ◽  
Wild Type ◽  
Conservation Analysis ◽  
Gene Assay ◽  
Protein Expressions ◽  
Patient Series ◽  
Reduced Activity

FGFR1 encodes a transmembrane cytokine receptor, which is involved in the early development of the human embryo and plays an important role in gastrulation, organ specification and patterning of various tissues. Pathogenic FGFR1 variants have been associated with Kallmann syndrome and hypogonadotropic hypogonadism. In our congenital scoliosis (CS) patient series of 424 sporadic CS patients under the framework of the Deciphering disorders Involving Scoliosis and COmorbidities (DISCO) study, we identified four unrelated patients harboring FGFR1 variants, including one frameshift and three missense variants. These variants were predicted to be deleterious by in silico prediction and conservation analysis. Signaling activities and expression levels of the mutated protein were evaluated in vitro and compared to that of the wild type (WT) FGFR1. As a result, the overall protein expressions of c.2334dupC, c.2339T>C and c.1261A>G were reduced to 43.9%, 63.4% and 77.4%, respectively. By the reporter gene assay, we observed significantly reduced activity for c.2334dupC, c.2339T>C and c.1261A>G, indicating the diminished FGFR1 signaling pathway. In conclusion, FGFR1 variants identified in our patients led to only mild disruption to protein function, caused milder skeletal and cardiac phenotypes than those reported previously.

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