Host proteostasis modulates influenza evolution

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance.National Institutes of Health (U.S.) (Award 1DP2GM119162)National Institutes of Health (U.S.) (Grant P30-ES002109

Analysis of ergonomics problems contact-centers

The analysis of the ergonomic problems of the contact center. It allocates main factors of influence on man- operator

Multidimensional chemical control of CRISPR–Cas9

Cas9-based technologies have transformed genome engineering and the interrogation of genomic functions, but methods to control such technologies across numerous dimensions-including dose, time, specificity, and mutually exclusive modulation of multiple genes-are still lacking. We conferred such multidimensional controls to diverse Cas9 systems by leveraging small-molecule-regulated protein degron domains. Application of our strategy to both Cas9-mediated genome editing and transcriptional activities opens new avenues for systematic genome interrogation

Characterization of an A-Site Selective Protein Disulfide Isomerase A1 Inhibitor

Protein disulfide isomerase A1 (PDIA1) is an endoplasmic reticulum (ER)-localized thiol-disulfide oxidoreductase that is an important folding catalyst for secretory pathway proteins. PDIA1 contains two active-site domains (a and a′), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. The two active-site domains share 37% sequence identity and function independently to perform disulfide-bond reduction, oxidation, and isomerization. Numerous inhibitors for PDIA1 have been reported, yet the selectivity of these inhibitors toward the a and a′ sites is poorly characterized. Here, we identify a potent and selective PDIA1 inhibitor, KSC-34, with 30-fold selectivity for the a site over the a′ site. KSC-34 displays time-dependent inhibition of PDIA1 reductase activity in vitro with a kinact/KI of 9.66 × 103 M-1 s-1 and is selective for PDIA1 over other members of the PDI family, and other cellular cysteine-containing proteins. We provide the first cellular characterization of an a-site selective PDIA1 inhibitor and demonstrate that KSC-34 has minimal sustained effects on the cellular unfolded protein response, indicating that a-site inhibition does not induce global protein folding-associated ER stress. KSC-34 treatment significantly decreases the rate of secretion of a destabilized, amyloidogenic antibody light chain, thereby minimizing pathogenic amyloidogenic extracellular proteins that rely on high PDIA1 activity for proper folding and secretion. Given the poor understanding of the contribution of each PDIA1 active site to the (patho)physiological functions of PDIA1, site selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1.National Institutes of Health (U.S.) (Grant 1R01AR071443

Directed evolution in mammalian cells

Directed evolution experiments are typically carried out using in vitro systems, bacteria, or yeast-even when the goal is to probe or modulate mammalian biology. Performing directed evolution in systems that do not match the intended mammalian environment severely constrains the scope and functionality of the targets that can be evolved. We review new platforms that are now making it possible to use the mammalian cell itself as the setting for directed evolution and present an overview of frontier challenges and high-impact targets for this approach.NSF Graduate Research Fellowship Program (Grant No. 1745302)NIH National Institute of General Medical Sciences (1R35GM136354

Chemical Biology Framework to Illuminate Proteostasis

© 2020 Annual Reviews Inc.. All rights reserved. Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis

Targeting defective proteostasis in the collagenopathies

© 2019 Elsevier Ltd The collagenopathies are a diverse group of diseases caused primarily by mutations in collagen genes. The resulting disruptions in collagen biogenesis can impair development, cause cellular dysfunction, and severely impact connective tissues. Most existing treatment options only address patient symptoms. Yet, while the disease-causing genes and proteins themselves are difficult to target, increasing evidence suggests that resculpting the intracellular proteostasis network, meaning the machineries responsible for producing and ensuring the integrity of collagen, could provide substantial benefit. We present a proteostasis-focused perspective on the collagenopathies, emphasizing progress toward understanding how mechanisms of collagen proteostasis are disrupted in disease. In parallel, we highlight recent advances in small molecule approaches to tune endoplasmic reticulum proteostasis that may prove useful in these disorders

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions In Vivo

Laboratory time scale evolution in vivo relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted in vivo mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for in vivo targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of in vivo evolution experiments.National Institutes of Health (Grant 1DP3DK094338–01)National Institutes of Health (Award P30-ES002109