Biology subjects

Bailey, A. O.

Publications and source records attributed to Bailey, A. O..

3 recordsLinked to original sources

bioRxiv2022

UNC-45 has a crucial role in maintaining muscle sarcomeres during aging in C. elegans

Summary/AbstractAs people live longer, age-related diseases, like sarcopenia, will become a greater public health concern. We use the model organism C. elegans to better understand the molecular mechanisms behind muscle maintenance. Muscle function is dependent on having properly organized and functioning thick filaments, which are primarily composed of myosin. The myosin head requires the chaperone UNC-45 to initially fold it after translation and is likely used to re-fold back to functionality after thermal or chemical stress induced unfolding. We observe an early onset of sarcopeania when UNC-45 is perturbed during adulthood. We observe that during adult aging, there is a sequential decline of HSP-90, UNC-45, and then myosin. Myosin and UNC-45 protein decline are independent of steady state mRNA levels. Loss of UNC-45 is correlated with an increase in phosphorylation of the protein. By mass spectrometry, S111 was identified as being phosphorylated and this modification may affect binding to HSP-90. A longevity mutant with delayed onset of sarcopenia also shows a delay in the loss of HSP-90, UNC-45, and myosin. We also see a decrease in UNC-45 protein, but not transcript, in an hsp-90 loss of function mutant, suggesting a role for HSP-90 in stabilizing UNC-45. This leads us to propose the model that during aging, a loss of HSP-90 leads to UNC-45 being post translationally modified, such as phosphorylation, and degraded, which then leads to a loss of myosin, and thus muscle mass and function. A better understanding of how myosin and its chaperone proteins are regulated and affected by aging will lead to better preventative care and treatment of sarcopenia and, possibly, the age-related decline of heart muscle function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/494828v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1040254org.highwire.dtl.DTLVardef@34f503org.highwire.dtl.DTLVardef@c1cd67org.highwire.dtl.DTLVardef@1945cf9_HPS_FORMAT_FIGEXP M_FIG C_FIG In young adults, under normal conditions the UCS domain of UNC-45 (shown in green) is bound to the myosin head (in red) and the TPR domain (in yellow) is bound to HSP-90 (in purple). Under stress conditions, HSP-90 detaches from the TPR domain, causing a conformational change in UNC-45 that allows the Central domain (in blue) to bind to the myosin neck (in red) resulting in inhibition of the myosin power stroke while the UCS domain protects/re-folds the myosin head. HSP-90 can then rebind the TPR domain, causing the Central domain to release the myosin neck, allowing movement of the myosin motor. However, aged adults experience a loss of HSP-90 and UNC-45 (which has increased post translational modification with aging). The loss of the Myosin chaperones leads to increased aggregation and degradation of Myosin with age.This loss of Myosin at the thick filament results in decline in muscle mass and function, also known as sarcopenia. Note that only the myosin head and neck are shown for simplicity of illustration.

cell biology↗

bioRxiv2022

Comparative genomic and crystal structure analyses identify a collagen glucosyltransferase from Acanthamoeba Polyphaga Mimivirus

Humans and Acanthamoeba Polyphaga Mimivirus share numerous homologous genes, including collagens and collagen-modifying enzymes. To explore the homology, we performed a genome-wide comparison between human and mimivirus using DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) and identified 52 new mimiviral proteins that are homologous with human proteins. To gain functional insights into mimiviral proteins, their human protein homologs were organized into Gene Ontology (GO) and REACTOME pathways to build a functional network. Collagen and collagen-modifying enzymes form the largest subnetwork with most nodes. Further analysis of this subnetwork identified a putative collagen glycosyltransferase R699. Protein expression test suggested that R699 is highly expressed in E coli, unlike the human collagen-modifying enzymes. Enzymatic activity assays showed that R699 catalyzes the conversion of unique galactosylhydroxylysine within the GXXXUG motif (U=galactosylhydroxylysine) to glucosylgalactosylhydroxylysine on collagen using uridine diphosphate glucose (UDP-Glc) as a sugar donor, suggesting R699 is a mimiviral collagen galactosylhydroxylysyl glucosyltransferase (GGT) with defined substrate specificity. Structural study of R699 produced the first crystal structure of a collagen GGT with a visible UDP-Glc. Sugar moiety of the UDP-Glc resides in a previously unrecognized pocket. Mn2+ coordination and nucleoside-diphosphate binding site are conserved among GGT family members and critical for R699s collagen GGT activity. To facilitate further analysis of human and mimiviral homologous proteins, we presented an interactive and searchable genome-wide comparison website for quickly browsing human and Acanthamoeba Polyphaga Mimivirus homologs, which is available at RRID Resource ID: SCR_022140 or https://guolab.shinyapps.io/app-mimivirus-publication/.

biochemistry↗

bioRxiv2021

Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 {+/-} 3% (total yield of purified protein: 270.5 {+/-} 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

molecular biology↗