Browsing by Author "Chhetri, Abhishek"

Now showing 1 - 2 of 2
  • Thumbnail Image
    ItemOpen Access
    Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway.
    (Biochemistry, 2018-01) Cox, Nathan J; Unlu, Gokhan; Bisnett, Brittany J; Meister, Thomas R; Condon, Brett M; Luo, Peter M; Smith, Timothy J; Hanna, Michael; Chhetri, Abhishek; Soderblom, Erik J; Audhya, Anjon; Knapik, Ela W; Boyce, Michael
    The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.
  • Thumbnail Image
    ItemOpen Access
    The Role of (1,3)-β-D-Glucan Synthase in Fungal Cell Wall Biosynthesis
    (2020) Chhetri, Abhishek

    (1,3)-β-D-glucan synthase is responsible for the biosynthesis of (1,3)-β-D-glucan, a

    major component of the fungal cell wall, and is a proven target of clinically approved

    antifungal antibiotics. Due to emerging drug resistance and the limited oral availabil-

    ity of existing drugs, there is growing interest in developing existing (1,3)-β-D-glucan

    synthase inhibitors as well as discovering new ones. However, development has been

    hampered by a limited understanding of the mechanisms of both the catalysis and

    inhibition of (1,3)-β-D-glucan synthase. This dissertation is an attempt to increase

    our understanding of (1,3)-β-D-glucan synthase catalysis and its role in fungal cell

    wall biosynthesis.

    I describe the development of a size exclusion chromatography-based method

    that allowed characterization of the lengths and amounts of (1,3)-β-D-glucan formed

    in assays, allowing the first functional and mechanistic characterization of (1,3)-β-

    D-glucan synthase. I also detail the synthesis and use of substrate analogs that

    provided strong support that (1,3)-β-D-glucan synthase elongates (1,3)-β-D-glucan

    chains from the non-reducing end, and does so processively with a turnover of ~0.5

    (1,3)-β-D-glucan min -1 . Based on the results, I propose a model for the catalytic

    mechanism of (1,3)-β-D-glucan synthase. The observation that glucanosyltransferase

    Gas1p is unnecessary for formation of (1,3)-β-D-glucan with biologically relevant

    degree of polymerization supports the model of fungal cell wall biosynthesis where

    (1,3)-β-D-glucan synthase is sufficient for generating (1,3)-β-D-glucan.

    ivAdditionally, I provide evidence that (1,3)-β-D-glucan synthase fractionates into

    detergent resistant membranes, and provide several strategies to overcome the chal-

    lenge of purifying (1,3)-β-D-glucan synthase for further biochemical studies. The

    techniques developed and reported here can also be applied to characterize other

    polysaccharide synthases.