Biology subjects

Misra, M.

Publications and source records attributed to Misra, M..

5 recordsLinked to original sources

bioRxiv2024

Development of covalent probes to capture Legionella pneumophila effector enzymes

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cells cytoplasm that hijack a plethora of cellular activities, including the hosts ubiquitination pathways. Effectors belonging to the SidE-family are involved in non-canonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires disease. This dynamic process is reversed by effectors called Dups that hydrolyse the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and x-ray crystallography approaches were used to identify the site of covalent crosslinking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

cell biology↗

bioRxiv2023

Biomimetic Lipid-polymer composite membrane model to study Permeability through Cornea

The excised animal cornea is the gold standard for testing and evaluating drug permeability through a cornea. However, it has a concise shelf life and encounters ethical concerns. Also, ex-vivo models, which are incomplete replicas of the human cornea, may provide faulty results in the pre-clinical studies. To circumvent these problems, we have proposed an in-vitro biomimetic lipid-polymer composite membrane (BLCM) model as an artificial cornea to study drug permeability. We designed and fabricated a free-standing, electro-spun polystyrene (PS) nanofibrous membrane and impregnated its pores with phosphatidylcholine (PC). SEM, FTIR, and goniometer characterized the BLCM. Permeation data of the drug ganciclovir through the BLCM model in a Franz-diffusion cell corroborates with the excised goat corneal system. Also, owing to the simple and scalable fabrication method, BLCM can be used as an alternative to animal models for initial drug permeability screening and studies and accelerate drug development.

bioengineering↗

bioRxiv2023

Identification of State Markers in Anorexia Nervosa: Replication and Extension of Inflammation Associated Biomarkers Using Multiplex Profiling in Anorexia Nervosa and Atypical Anorexia Nervosa

Proteomics provides an opportunity for detection and monitoring of anorexia nervosa (AN) and its related variant, atypical-AN (atyp-AN). However, research to date has been limited by the small number of proteins explored, exclusive focus on adults with AN, and lack of replication across studies. This study performed Olink Proseek Multiplex profiling of 92 proteins involved in inflammation among females with AN and atyp-AN (N = 64), all < 90% of expected body weight, and age-matched healthy controls (HC; N=44). After correction for multiple testing, nine proteins differed significantly in the AN/atyp-AN group relative to HC group (lower levels: CXCL1, HGF, IL-18R1, TNFSF14, TRANCE; higher levels: CCL23, Flt3L, LIF-R, MMP-1). The expression levels of three proteins (lower IL-18R1, TRANCE; higher LIF-R) were uniquely disrupted in females with AN. No unique expression levels emerged for atyp-AN. Across the whole sample, twenty-one proteins correlated positively with BMI (ADA, AXIN1, CD5, CD244, CD40, CD6, CXCL1, FGF-21, HGF, IL-10RB, IL-12B, IL18, IL-18R1, IL6, LAP TGF-beta-1, SIRT2, STAMBP, TNFRSF9, TNFSF14, TRAIL, TRANCE) and six (CCL11, CCL23, FGF-19, IL8, LIF-R, OPG) were negatively correlated with BMI. Overall, our results replicate the prior study demonstrating a dysregulated inflammatory status in AN, and extend these results to atyp-AN (AN/atyp-AN all < 90% of expected body weight). Of the 27 proteins correlated with BMI, 18 were replicated from a prior study using similar methods, highlighting the promise of inflammatory protein expression levels as biomarkers of disease monitoring. Additional studies of individuals across the entire weight spectrum are needed to understand the role of inflammation in atyp-AN.

immunology↗

bioRxiv2023

Serine ubiquitination of p62 regulates Nrf2 dependent redox homeostasis

The KEAP1-Nrf2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of Cullin-3 ubiquitin ligase that controls the cellular levels of Nrf2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate Nrf2 signaling through phosphorylation of the adaptor protein p62, which competes with Nrf2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of p62 catalyzed by SidE effectors of Legionella pneumophila controls Nrf2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of p62 sterically blocks its binding to KEAP1, resulting in Nrf2 ubiquitination and degradation. This reduces Nrf2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated p62 diminish in the later stage of infection allowing the expression of Nrf2-target genes; resulting in a differential regulation of the host metabolome and proteome in a Nrf2 dependent manner.

cell biology↗

bioRxiv2023

Mechanism and evolutionary origins of Alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10

In Ribosome-associated Quality Control (RQC), nascent-polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ( Ala-tails) that function outside ribosomes to induce ubiquitylation by Pirh2 or CRL2-KLHDC10 E3 ligases. Here we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails, and structural predictions identify candidate Ala-tail binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.

molecular biology↗