Herpesviruses, which cause many incurable diseases, infect cells by fusing viral and cellular membranes. While most other enveloped viruses use a single viral catalyst, called a fusogen, herpesviruses, inexplicably, require two conserved fusion-machinery components, gB and the heterodimer gH–gL, plus other non-conserved components. gB is a class III viral fusogen but, unlike other members of its class, does not function alone. We determined the crystal structure of the gH ectodomain bound to gL, from herpes simplex virus 2. gH–gL is an unusually tight complex of a novel architecture that, unexpectedly, does not resemble any known viral fusogen. Instead, we propose that gH–gL activates gB for fusion, possibly through direct binding. Formation of a gB–gH–gL complex is critical for fusion and is inhibited by a neutralizing antibody, making gB–gH–gL interface a promising antiviral target.
A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.
The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified 5 different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib compared to native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the ‘gatekeeper’ mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.
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