Construction of a Highly Sensitive Thiol-Reactive AIEgen-Peptide Conjugate for Monitoring Protein Unfolding and Aggregation in Cells.

Impairment of the protein quality control network leads to the accumulation of unfolded and aggregated proteins. Direct detection of unfolded protein accumulation in the cells may provide the possibility for early diagnosis of neurodegenerative diseases. Here a new platform based on a peptide-conjugated thiol-reactive aggregation-induced emission fluorogen (AIEgen), named MI-BTD-P (or D1), for labeling and tracking unfolded proteins in cells is reported. In vitro experiments with model proteins show that the non-fluorescent D1 only becomes highly fluorescent when reacted with the thiol group of free cysteine (Cys) residues on unfolded proteins but not glutathione or folded proteins with buried or surface exposed Cys. When the labeled unfolded proteins form aggregates, D1 fluorescence intensity is further increased, and fluorescence lifetime is prolonged. D1 is then used to measure unfolded protein loads in cells by flow cytometry and track the aggregate formation of the D1 labeled unfolded proteins using confocal microscopy. In combination with fluorescence lifetime imaging technique, the proteome at different folding statuses can be better differentiated, demonstrating the versatility of this new platform. The rational design of D1 demonstrates the outlook of incorporation of diverse functional groups to achieve maximal sensitivity and selectivity in biological samples.

Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates.

The disulfide bond (DSB) forming system and in particular DsbA, is a key bacterial oxidative folding catalyst. Due to its role in promoting the correct assembly of a wide range of virulence factors required at different stages of the infection process, DsbA is a master virulence rheostat, making it an attractive target for the development of new virulence blockers. Although DSB systems have been extensively studied across different bacterial species, to date, little is known about how DsbA oxidoreductases are able to recognize and interact with such a wide range of substrates. This review summarizes the current knowledge on the DsbA enzymes, with special attention on their interaction with the partner oxidase DsbB and substrates associated with bacterial virulence. The structurally and functionally diverse set of bacterial proteins that rely on DsbA-mediated disulfide bond formation are summarized. Local sequence and secondary structure elements of these substrates are analyzed to identify common elements recognized by DsbA enzymes. This not only provides information on protein folding systems in bacteria but also offers tools for identifying new DsbA substrates and informs current efforts aimed at developing DsbA targeted anti-microbials.

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Impact of COVID-19 pandemic on socioeconomic and mental health aspects in Nepal.

BACKGROUND: Public health concern is increasing with recent rise in the number of COVID-19 cases in Nepal. To curb this pandemic, Nepal is facing some forms of lockdown, encouraging people to implement social distancing so as to reduce interactions between people which could eventually reduce the possibilities of new infection; however, it has affected the overall physical, mental, social and spiritual health of the people. METHODS: Published articles related to psychosocial effects due to COVID-19 and other outbreaks were searched and reviewed. CONCLUSION: While many countries are supporting their citizens with sophisticated health safety-nets and various relief funds, some developing countries have unique challenges with vulnerable populations and limited resources to respond to the pandemic. This review presents the consequences of pandemic and lockdown on socioeconomic, mental health and other aspects in Nepalese society.

A Molecular Chameleon for Mapping Subcellular Polarity in an Unfolded Proteome Environment.

Environmental polarity is an important factor that drives biomolecular interactions to regulate cell function. Herein, a general method of using the fluorogenic probe NTPAN-MI is reported to quantify the subcellular polarity change in response to protein unfolding. NTPAN-MI fluorescence is selectively activated upon labeling unfolded proteins with exposed thiols, thereby reporting on the extent of proteostasis. NTPAN-MI also reveals the collapse of the host proteome caused by influenza A virus infection. The emission profile of NTPAN-MI contains information of the local polarity of the unfolded proteome, which can be resolved through spectral phasor analysis. Under stress conditions that disrupt different checkpoints of protein quality control, distinct patterns of dielectric constant distribution in the cytoplasm can be observed. However, in the nucleus, the unfolded proteome was found to experience a more hydrophilic environment across all the stress conditions, indicating the central role of nucleus in the stress response process.

The Scs disulfide reductase system cooperates with the metallochaperone CueP in Salmonella copper resistance.

The human pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) contains a complex disulfide bond (Dsb) catalytic machinery. This machinery encompasses multiple Dsb thiol-disulfide oxidoreductases that mediate oxidative protein folding and a less-characterized suppressor of copper sensitivity (scs) gene cluster, associated with increased tolerance to copper. To better understand the function of the Salmonella Scs system, here we characterized two of its key components, the membrane protein ScsB and the periplasmic protein ScsC. Our results revealed that these two proteins form a redox pair in which the electron transfer from the periplasmic domain of ScsB (n-ScsB) to ScsC is thermodynamically driven. We also demonstrate that the Scs reducing pathway remains separate from the Dsb oxidizing pathways and thereby avoids futile redox cycles. Additionally, we provide new insight into the molecular mechanism underlying Scs-mediated copper tolerance in Salmonella We show that both ScsB and ScsC can bind toxic copper(I) with femtomolar affinities and transfer it to the periplasmic copper metallochaperone CueP. Our results indicate that the Salmonella Scs machinery has evolved a dual mode of action, capable of transferring reducing power to the oxidizing periplasm and protecting against copper stress by cooperating with the cue regulon, a major copper resistance mechanism in Salmonella. Overall, these findings expand our understanding of the functional diversity of Dsb-like systems, ranging from those mediating oxidative folding of proteins required for infection to those contributing to defense mechanisms against oxidative stress and copper toxicity, critical traits for niche adaptation and survival.

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens.

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against Neisseria-specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among Neisseria spp. and that this enzyme is essential for survival of N. gonorrhoeae DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of Neisseria DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the Neisseria n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.

Autotransporter Adhesins in Escherichia coli Pathogenesis.

Most bacteria produce adhesion molecules to facilitate the interaction with host cells and establish successful infections. An important group of bacterial adhesins belong to the autotransporter (AT) superfamily, the largest group of secreted and outer membrane proteins in Gram-negative bacteria. AT adhesins possess diverse functions that facilitate bacterial colonisation, survival and persistence, and as such are often associated with increased bacterial fitness and pathogenic potential. In this review, we will describe AIDA-I type AT adhesins, which comprise the biggest and most diverse group in the AT family. We will focus on Escherichia coli proteins and define general aspects of their biogenesis, distribution, structural properties and key roles in infection.