Biology subjects

Raghu, P.

Publications and source records attributed to Raghu, P..

7 recordsLinked to original sources

bioRxiv2022

Structural rationale to understand the effect of disease-associated mutations on Myotubularin

Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The MTM1 gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains- a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerization of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed in silico and in vitro studies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature. Author SummaryX-linked myotubular myopathy is a rare paediatric disorder and affected males suffer neonatal death or may live on only with ventilatory support. In this study, we employed a range of approaches to understand the molecular level effects of patient-derived mutations on an enzyme directly linked to the congenital muscular disorder. Using three-dimensional modelling and simulations, the effect of these mutations on the structure of the enzyme and its ability to bind its substrate was studied. To complement theoretical observations, experiments were performed with cells expressing this enzyme and its mutants. These studies reveal that each part of the protein may directly or indirectly affect its enzyme activity and most of the patient-derived mutations are expressed insufficiently in the cell. With the advent of genome sequencing technology, identification of congenital mutations is easier; computational studies of molecular consequences of mutations on a protein function such as this will prove immensely useful in understanding the disease and its prognosis.

bioinformatics↗

bioRxiv2020

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

During phospholipase C-{beta} (PLC-{beta}) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.

cell biology↗

bioRxiv2019

Extended synaptotagmin regulates plasma membrane-endoplasmic reticulum contact site structure and lipid transfer function in vivo

Inter-organelle communication between closely apposed membranes is proposed at Membrane Contact Sites (MCS). However the regulation of MCS structure and their functional relevance in vivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. We report that in Drosophila photoreceptors, MCS number is regulated by PLC{beta} activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER-PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP2 resynthesis seen in rdgB mutants. Thus, dEsyt function and PLC{beta} signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.

cell biology↗

bioRxiv2019

The lipid transfer function of RDGB at ER-PM contact sites is regulated by multiple interdomain interactions.

In Drosophila photoreceptors, following Phospholipase C-{beta} activation, the phosphatidylinositol transfer protein (PITP) RDGB, is required to maintain lipid homeostasis at endoplasmic reticulum (ER) plasma membrane (PM) membrane contact sites (MCS). Depletion or mis-localization of RDGB results in multiple defects in photoreceptors. Previously, interaction between the FFAT motif of RDGB with the integral ER protein dVAP-A was shown to be important for its localization at ER-PM MCS. Here, we report that in addition to FFAT motif, a large unstructured region (USR1) of RDGB is required to support the RDGB/dVAP-A interaction. However, interaction with dVAP-A alone is insufficient for accurate localization of RDGB: this also requires association of RDGB with apical PM, through its C-terminal LNS2 domain. Deletion of LNS2 domain results in complete mis-localisation of RDGB and also induces large mis-regulated interdomain movements abrogating RDGB function. Thus, multiple independent interactions between individual domains of RDGB supports its function at ER-PM MCS.

cell biology↗

bioRxiv2019

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In this study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use 18O-ATP to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from both Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity levels from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

biochemistry↗

bioRxiv2018

Insulin sensitivity and PIP3 turnover in Drosophila are regulated by phosphatidylinositol 5 phosphate 4-kinase.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation at the plasma membrane is a key event during activation of receptor tyrosine kinases such as the insulin receptor and is critical for normal growth and metabolism. The lipid kinases and phosphatases regulating PIP3 levels are described but mechanisms controlling their activity remain unclear. We report that in Drosophila, phosphatidylinositol 5 phosphate 4-kinase (PIP4K) function at the plasma membrane is required for normal PIP3 levels during insulin receptor activation. Depletion of PIP4K increases PIP3 levels and augments sensitivity to insulin through enhanced Class I phosphoinositide 3-kinase (PI3K) activity. Animals lacking PIP4K show enhanced insulin signalling dependent phenotypes in vivo and are resistant to the metabolic consequences of a high-sugar diet, highlighting the importance of PIP4K in normal metabolism and development. Thus, PIP4KS are key regulators of receptor tyrosine kinase signalling with implications for growth factor dependent processes including tumour growth, T-cell activation and metabolism.

cell biology↗

bioRxiv2017

Evidence of sinks and sources in the PLC activated PIP2 cycle

In many eukaryotic signalling cascades, receptor mediated phospholipase C (PLC) activity triggers phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis leading to information transfer in cells. Coupled with PLC activation is a sequence of reactions spread across multiple compartments by which PIP2 is resynthesized, a process essential to maintain PIP2 levels and support sustained PLC signalling. The biochemical strategies to co-ordinate these reactions and support PIP2 levels have remained poorly understood. In particular, the question of whether the PIP2 cycle is a closed cycle with no net addition or loss of metabolites has not been addressed. Using mathematical modelling approaches, we find that a closed PIP2 cycle cannot explain experimentally observed changes in the metabolic intermediates when changing enzyme activities in the PIP2 cycle. Thus, we propose that the PIP2 cycle likely includes at least one metabolic source and one sink whose net activity results in the experimentally observed regulation of this key signalling pathway.

biophysics↗