Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to +/- 80 nm, extending the protein’s light absorption significantly beyond the range of known natural PPRs. While proton pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurrred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty-four of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the wild-type protein

Effectiveness of a computer assisted learning (CAL) package to raise awareness of autism

<p>Abstract</p> <p>Background</p> <p>Promoting awareness of autism in populations who work with children may result in an earlier diagnosis of the condition. In this study, a computer assisted learning (CAL) package, containing educationally appropriate knowledge about autism was developed; and the effectiveness of this CAL package was evaluated.</p> <p>Methods</p> <p>The CAL package was developed using computer software, "Xerte" and "Flash Macromedia". The effectiveness of the CAL package was evaluated in 32 childcare students in the UK, who were randomised to watch the CAL package or to read the information leaflet containing the same information (n = 16 in each group). Retention performance, level of enjoyment, and level of confidence to identify a child with autism, after the interventions, were evaluated. The data obtained from two studied groups was analysed using unpaired Student's t-test, 95% confidence interval, and effect size.</p> <p>Results</p> <p>Students who watched the CAL package had superior retention performance percentage scores (p = 0.02, 95% CI = 0.83–12.19, effect size = 0.8) and level of enjoyment (p = 0.04, 95% CI = 0.03–2.75, effect size = 0.7) compared with students who read the information leaflet. However, there was no significant difference in level of confidence to identify a child with autism (p = 0.39, 95% CI = -1.80–0.72, effect size = -0.3).</p> <p>Conclusion</p> <p>The CAL package developed was an effective method of educating people who work with children about autism.</p

Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy

BACKGROUND:Mesenchymal progenitor cells (MPCs) have been isolated from a variety of connective tissues, and are commonly called "mesenchymal stem cells" (MSCs). A stem cell is defined as having robust clonal self-renewal and multilineage differentiation potential. Accordingly, the term "MSC" has been criticised, as there is little data demonstrating self-renewal of definitive single-cell-derived (SCD) clonal populations from a mesenchymal cell source. METHODOLOGY/PRINCIPAL FINDINGS:Here we show that a tractable MPC population, human umbilical cord perivascular cells (HUCPVCs), was capable of multilineage differentiation in vitro and, more importantly, contributed to rapid connective tissue healing in vivo by producing bone, cartilage and fibrous stroma. Furthermore, HUCPVCs exhibit a high clonogenic frequency, allowing us to isolate definitive SCD parent and daughter clones from mixed gender suspensions as determined by Y-chromosome fluorescent in situ hybridization. CONCLUSIONS/SIGNIFICANCE:Analysis of the multilineage differentiation capacity of SCD parent clones and daughter clones enabled us to formulate a new hierarchical schema for MSC self-renewal and differentiation in which a self-renewing multipotent MSC gives rise to more restricted self-renewing progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached

MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment

Summary: The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design. : The mechanisms that regulate intracellular viral envelopment are unknown. Kumar et al. report that MARCH8 catalyzes K63-linked polyubiquitination of the HCV nonstructural 2 protein and subsequently ESCRT recruitment and HCV envelopment. MARCH8 is required for infection with other Flaviviridae family members, thereby representing a potential host target for antiviral strategies. Keywords: ubiquitination, hepatitis C virus, HCV, MARCH8, virus-host interactions, proteomics, assembly, envelopment, intracellular membrane trafficking, ESCR

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses

Optimization of Isothiazolo[4,3-b]pyridine-Based Inhibitors of Cyclin G Associated Kinase (GAK) with Broad-Spectrum Antiviral Activity

There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo[4,3- b]pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo[4,3- b]pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections.status: publishe

Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release

Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread

The cargo adaptor protein CLINT1 is phosphorylated by the Numb-associated kinase BIKE and mediates dengue virus infection

International audienceThe signaling pathways and cellular functions regulated by the four Numb-associated kinases (NAKs) are largely unknown. We previously reported that AAK1 and GAK control intracellular trafficking of RNA viruses, and recently revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE in this process have not yet been identified. Here, to identify BIKE substrates, we conducted a barcode fusion genetics-yeast two-hybrid screen and retrieved publicly available data generated via affinity-purification mass spectrometry. We subsequently validated 19 of 47 putative BIKE interactors using mammalian cell-based protein-protein interaction assays. We found that CLINT1, a cargo-specific adaptor implicated in bidirectional Golgi-to-endosome trafficking, emerged as a predominant hit in both screens. Our experiments indicated that BIKE catalyzes phosphorylation of a threonine 294 (T294) CLINT1 residue both in vitro and in cell culture. Our findings revealed that CLINT1 phosphorylation mediates its binding to the DENV nonstructural 3 protein and subsequently promotes DENV assembly and egress. In addition, using live-cell imaging we revealed that CLINT1 cotraffics with DENV particles and is involved in mediating BIKE's role in DENV infection. Finally, our data suggest that additional cellular BIKE interactors implicated in the host immune and stress responses and the ubiquitin proteasome system might also be candidate phosphorylation substrates of BIKE. In conclusion, these findings reveal cellular substrates and pathways regulated by the understudied NAK enzyme BIKE, a mechanism for CLINT1 regulation, and control of DENV infection via BIKE signaling, with potential implications for cell biology, virology, and host-targeted antiviral design

Optimization of Isothiazolo[4,3‑<i>b</i>]­pyridine-Based Inhibitors of Cyclin G Associated Kinase (GAK) with Broad-Spectrum Antiviral Activity

There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo­[4,3-<i>b</i>]­pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo­[4,3-<i>b</i>]­pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections