Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 Å from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal β-hairpin at the distal site-a surface-exposed hydrophobic crevice 17 Å away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 Å of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.deubiquitinating enzyme | enzyme suicide substrate complex | neurodegeneration | ubiquitination U CHL1, a member of the UCH (ubiquitin C-terminal hydrolase) family of deubiquitinating enzymes (DUBs), is a 223-amino acid protein found abundantly and selectively expressed in brain, constituting up to 1-2% of total brain protein (1, 2). In vivo studies suggest that UCHL1 is involved in regulation of ubiquitin pool, apoptosis, and learning and memory, and its absence in mice because of spontaneous intragenic deletions yields phenotypes with neurological defects (3). Mutations in UCHL1 have been implicated in Parkinson disease (PD). A point mutation near the active site that changes Ile93 to Met (I93M) has been linked to an increased risk of developing an autosomaldominant form of PD (4). Conversely, a common S18Y polymorphism reduces susceptibility to PD (5, 6) and Alzheimer's disease (7). In addition to its association with neurodegenerative diseases, abnormal expression of UCHL1 is found in many forms of cancer, including lung, colorectal, and pancreatic cancers, and may be related to tumor progression (8, 9). The normal function of UCHL1, however, is not known. Also unknown are how the activity of this abundant neuronal enzyme is regulated and what its true physiological substrates are, although biochemical studies have indicated that UCHL1 can accept short-peptide (α or ϵ-amino-linked) or small-molecule C-terminal conjugates of ubiquitin as substrates, cleaving, as its name suggests, the amide bond following immediately after the C-terminal glycine (Gly7...

(-)-Epigallocatechin-3-gallate (EGCG), the major constituent of green tea has been reported to prevent many diseases by virtue of its antioxidant properties. The binding of EGCG with human serum albumin (HSA) has been investigated for the first time by using fluorescence, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and protein-ligand docking. We observed a quenching of fluorescence of HSA in the presence of EGCG. The binding parameters were determined by a Scatchard plot and the results were found to be consistent with those obtained from a modified Stern-Volmer equation. From the thermodynamic parameters calculated according to the van't Hoff equation, the enthalpy change deltaH degrees and entropy change deltaS degrees were found to be -22.59 and 16.23 J/mol K, respectively. These values suggest that apart from an initial hydrophobic association, the complex is held together by van der Waals interactions and hydrogen bonding. Data obtained by fluorescence spectroscopy, CD, and FTIR experiments along with the docking studies suggest that EGCG binds to residues located in subdomains IIa and IIIa of HSA. Specific interactions are observed with residues Trp 214, Arg 218, Gln 221, Asn 295 and Asp 451. We have also looked at changes in the accessible surface area of the interacting residues on binding EGCG for a better understanding of the interaction.

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae, the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae. Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens. cholera | antimicrobial resistance | mobile genetic elements | genome | proteome

Protein post-translational modification by ubiquitin represents a complex signaling system that regulates many cellular events including proteostasis to intercellular communications. Deubiquitinating enzymes (DUBs) that specifically disassemble Ub-chains or regulate ubiquitin homeostasis reside as a central component in ubiquitin signaling. Human genome encodes almost 100 DUBs and majority of them are not well characterized. Considerable progress has been made in the understanding of enzymatic mechanism; however, their cellular substrate specificity and regulation are largely unknown. Involvement of DUBs in disease regulation has been depicted since its discovery and several attempts have been made for evaluating DUBs as a drug target. In this review, we have updated briefly a new insight of DUBs activity, their cellular role, disease regulation, and therapeutic potential. V C 2015 IUBMB Life, 67(7): [544][545][546][547][548][549][550][551][552][553][554][555] 2015

Microfilarial protein appears to be a new ligand of TLR4 from W. bancrofti. Determination of its functional attributions in the host-parasite relationship, especially immunopathogenesis of filarial infection, may improve our understanding.

Ubiquitin C-terminal Hydrolase-1 (UCHL1) is a deubiquitinating enzyme, which plays a key role in Parkinson’s disease (PD). It is one of the most important proteins, which constitute Lewy body in PD patient. However, how this well folded highly soluble protein presents in this proteinaceous aggregate is still unclear. We report here that UCHL1 undergoes S-nitrosylation in vitro and rotenone induced PD mouse model. The preferential nitrosylation in the Cys 90, Cys 152 and Cys 220 has been observed which alters the catalytic activity and structural stability. We show here that nitrosylation induces structural instability and produces amorphous aggregate, which provides a nucleation to the native α-synuclein for faster aggregation. Our findings provide a new link between UCHL1-nitrosylation and PD pathology.

Background: Atypical hemolytic uremic syndrome (aHUS), an important cause of acute kidney injury (AKI), is characterized by dysregulation of the alternative complement pathway. Autoantibodies to factor H (FH), a chief regulator of this pathway, account for a distinct subgroup. While high anti-FH titers predict relapse, they do not correlate well with disease activity and their functional characterization is required. Methods: Of 781 patients <18-year-old of aHUS in the nationwide database from 2007 to 2018, 436 (55.8%) had anti-FH antibodies. Clinical features and outcome of patients managed in the last 6-year ( n = 317) were compared to before ( n = 119). In plasma samples of 44 patients, levels of serial circulating FH immune complexes (CIC), free FH, soluble terminal complement complex (sC5b-9), sheep red blood cell (SRBC) lysis and epitope specificity ( n = 8) were examined. Functional renal reserve, ambulatory hypertension, left ventricular hypertrophy (LVH), and proteinuria were evaluated in a subset. Results: Patients presented with markedly elevated anti-FH titers (10,633.2 ± 998.5 AU/ml). Management varied by center, comprising plasma exchange (PEX; 77.5%) and immunosuppression (73.9%). Patients managed in the last 6-year showed better renal survival at mean 28.5 ± 27.3 months (log rank P = 0.022). Mean anti-FH titers stayed 700–1,164 AU/ml during prolonged follow-up, correlating with CIC. Patients with relapse had lower free-FH during remission [Generalized estimating equations (GEE), P = 0.001]; anti-FH levels ≥1,330 AU/ml and free FH ≤440 mg/l predicted relapse (hazards ratio, HR 6.3; P = 0.018). Epitope specificity was similar during onset, remission and relapse. Antibody titer ≥8,000 AU/ml (HR 2.23; P = 0.024), time to PEX ≥14 days (HR 2.09; P = 0.071) and PEX for <14 days (HR 2.60; P = 0.017) predicted adverse renal outcomes. Combined PEX and immunosuppression improved long-term outcomes (HR 0.37; P = 0.026); maintenance therapy reduced risk of relapses (HR 0.11; P < 0.001). At 4.4±2.5 year, median renal reserve was 15.9%; severe ambulatory, masked and pre-hypertension were found in 38, 30, and 18%, respectively. Proteinuria and LVH occurred in 58 and 28% patients, respectively. Conclusion: Prompt recognition and therapy with PEX and immunosuppression, is associated with satisfactory outcomes. Free-FH predicts early relapses in patients with high anti-FH titers. A significant proportion of impaired functional reserve, ambulatory hypertension, proteinuria and LVH highlight the need for vigilant long-term follow-up.

In an attempt to screen out cellulase producing bacteria from herbivorous animal fecal matter it was possible to isolate a potent bacterium from cow dung. The bacterium was identified as Bacillus sp. using 16S rDNA based molecular phylogenetic approach. The effect of different agricultural wastes, paper wastes and carboxymethyl cellulose on endoglucanase production was tested and was found to produce maximally at 8% carboxymethyl cellulose. The endoglucanase was precipitated by ammonium sulfate saturation and purified by DEAE- Sepharose column. The purification was achieved 8.5 fold from the crude extract with a yield of 68.1%. The molecular weight of the protein was determined to be 97 kDa by SDS-PAGE. The enzymatic activity was moderately reduced by detergents (SDS, Tween-80), metal ions (MnCl2, ZnCl2) and EDTA. The endoglucanase was stable between pH 5.0 – 9.0 and temperature between 20−70°C with optimal activity at pH 7.0 and temperature 50°C. The apparent Km value of the enzyme for the substrate carboxymethyl cellulose was recorded to be 0.25 mg/ml. The endoglucanase was stable in the presence of commercial detergents such as Ariel, Surf Excel and Tide, indicated might be of potential applications in detergent industry. The enzyme from this strain could also be applied in bioconversion of lignocellulosic biomass into fermentable sugars.