Biology subjects

Diril, M. K.

Publications and source records attributed to Diril, M. K..

3 recordsLinked to original sources

bioRxiv2026

Identification of Kinome Biomarkers and Therapeutic Rescue in cGMP-Linked Retinal Degeneration

Inherited retinal degenerations, such as Retinitis Pigmentosa (RP) and Leber's congenital amaurosis (LCA), are major causes of progressive and irreversible vision loss. The large number of genes associated with these disorders makes their clinical and molecular characterization difficult, while systemic biomarkers for disease progression or treatment response remain limited. These challenges were addressed by targeting cGMP-associated mechanisms shared by RP and LCA and integrating systemic biomarker profiling with retinal outcomes. Kinase activity analysis (kinome analysis) in peripheral blood mononuclear cells distinguished healthy and diseased states and captured therapy-associated signatures in rd10 mice treated with LP-CN03, a liposomal formulation of the cGMP analog CN03. This first systemic analysis in a therapeutically responsive animal model highlights kinome analysis as a potential translational readout. Given shared cGMP signaling in RP and AIPL1-associated LCA, LP-CN03's therapeutic potential was further evaluated in a new CRISPR-Cas9-generated Aipl1-/- mouse model. Treatment with LP-CN03 preserved retinal structure and function, as shown by electroretinography, longitudinal optical coherence tomography, fundus imaging, optomotor testing, and photoreceptor-specific gene expression. Together, these findings provide independent in vivo evidence for the neuroprotective effect of LP-CN03 in both cGMP-related RP and LCA, also highlighting the translational value of kinome profiling.

neuroscience↗

bioRxiv2024

Characterization of a novel neurodevelopmental rare disease caused by a mutation within the autophagy gene ATG9B

Autophagy is a highly conserved eukaryotic cellular process whose dysfunction results in human pathologies including cancer and neurodegenerative disease. First identified in yeast, ATG genes are central players in autophagy. Although their roles in cancer and neurodegenerative disease are well known, Mendelian diseases associated with ATG genes are rare. Mutations in core autophagy genes ATG5 and ATG7 have been previously reported to cause rare genetic disorders with autosomal recessive inheritance pattern. Here we report, for the first time, a rare genetic disorder that results from a deletion/frameshift mutation in human ATG9B, the placenta specific homologue of yeast ATG9 in humans. The 11-nucleotide deletion causes a frameshift and addition of a premature stop codon, truncating the C-terminal cytosolic domain of the ATG9B protein. The pediatric patients carrying the mutant allele homozygous were children of a consanguineous marriage and displayed neurodevelopmental anomalies including mental retardation. We hypothesized that this phenotype originates during placental development. To characterize the effects of the mutation and gain insight on the specific functions of ATG9B in a physiological setting, we used mammalian cells and generated a knock-in mouse model. Truncated ATG9B was not stable when expressed in cells. It was localized to perinuclear vesicles like the WT protein, but not to peripheral vesicles. Homozygous knock-in mice were viable, fertile and displayed no gross phenotypical abnormalities. Histomorphometry analysis of the placenta layers did not reveal a significant difference between mutant and control embryos. The assessments of neurobehavioral tests were similar in wild-type and homozygous knock-in mice. However, knock-in mice had a reduced fear memory trend, which is an amygdala-involved response.

genetics↗

bioRxiv2020

Investigating the determinants of mammalianMastl kinase activation

Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of the AGC kinase family, possessing a unique non-conserved middle region (NCMR). The mechanism of its activation prior to mitosis has been extensively studied in Xenopus egg extracts. These studies found several residues (corresponding to T193 and T206 in the activation loop, and S861 in the C-terminal tail, i.e., C-tail of mouse Mastl) whose phosphorylations are crucial for enzymatic activation. To date, the significance of these phosphosites was not confirmed in live mammalian cells. Here, we utilize a complementation cloning approach to determine the essentials of mammalian Mastl kinase activity. We employed a tamoxifen-inducible conditional knockout system to delete the endogenous Mastl in mouse embryonic fibroblasts (MEF) and screened various mutants for their ability to complement its loss. MEFs, ectopically expressing different phosphorylation site mutants, were induced to undergo recombination-mediated knockout in their endogenous Mastl loci. S861A and S861D mutants were able to complement endogenous Mastl loss with proliferation rates comparable to WT. In parallel, we examined the available protein kinase structures having a phosphorylated C-tail. Among the published states, two distinct positionings of the C-tail phosphoresidue were observed. Energetic analysis of these states revealed that only one conformation highly contributes to the C-tail docking. Our in-depth sequence and structure analysis showed that Mastl pS861 does not belong to the conformational state, where the phosphoresidue contributes to the C-tail docking. The C-tail of Mastl is relatively short and it lacks the hydrophobic (HF) motif. In other AGC kinases, the C-tail phosphosite aids the anchoring of this motif over the N-lobe, leading to the final step of kinase activation. Together with the lack of HF motif in Mastl, our results suggest that phosphorylation of the C-tail turn motif phosphosite (S861) is auxiliary and is dispensable for mammalian Mastl kinase function. Furthermore, we demonstrated that complementation cloning is a powerful approach for screening the determinants of an essential proteins functioning.

cell biology↗