Biology subjects

Kanyo, J.

Publications and source records attributed to Kanyo, J..

4 recordsLinked to original sources

bioRxiv2021

A versatile new ubiquitin detection and purification tool derived from a bacterial deubiquitylase

Protein ubiquitylation is an important post-translational modification affecting an wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on developing methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain derived from an Orientia tsutsugamushi deubiquitylase. We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As a proof-of-principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin researchers with specific advantages over other methods, such as in detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

biochemistry↗

bioRxiv2021

A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and while its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo, using developing spermatogenic cells as a model. Remarkably, we find that AGO2 acts in the germ cell nucleus to positively regulate protein expression. We show that AGO2 dynamically binds both chromatin and nuclear mRNA transcripts of hundreds of genes required for sperm production, and germline conditional knockout (cKO) of Ago2 causes depletion of the corresponding proteins, along with defects in sperm number and morphology. Nuclear AGO2 partners with splicing, export, and chromatin factors to promote transcript export and protein expression. Together, our data reveal an unexpected role for nuclear AGO2 in enhancing expression of developmentally important genes.

developmental biology↗

bioRxiv2021

Carbonyl Post-Translational Modification Associated with Early Onset Type 1 Diabetes Autoimmunity

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various post-translational modifications (PTMs) to self-proteins. Herein, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in non-obese diabetic (NOD) mice. Of particular interest, we identified carbonyl modification of the prolyl-4-hydroxylase beta subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found the carbonylated-P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin to insulin ratios. Moreover, circulating autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and those creating autoantigenic forms of insulin in type 1 diabetes.

immunology↗

bioRxiv2021

Citrullination of glucokinase linked to autoimmune diabetes

Inflammation, including reactive oxygen species and inflammatory cytokines in tissue microenvironments amplify the appearance of various post-translational modifications (PTMs) of self-proteins. Previously, a number of PTMs have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes (T1D). Herein, we have identified the citrullination of glucokinase (GK) as a result of inflammation, triggering autoimmunity and affecting its biological functions. Glucokinase is predominantly expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells, where it acts as a glucose sensor to initiate glycolysis and insulin signaling. Herein, we demonstrate that glucokinase is citrullinated in inflamed non-obese diabetic (NOD) islets as well as in human GK. Autoantibodies against both native and citrullinated GK arise in both spontaneous human T1D and murine models. Likewise, autoreactive CD4+ T cells to both native and citrullinated glucokinase epitopes are present in the circulation of T1D patients. Finally, citrullination alters GK biologic activity and suppresses glucose-stimulated insulin secretion. Our studies define glucokinase as a T1D biomarker, providing new insights into altering glucose metabolism, creating neoautoantigens, and better define the broad impact of PTMs on the tissue pathology of T1D.

immunology↗