👤 Dario R Alessi

Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinson's disease. Here we show that VPS13C, a bridge-like lipid-transport protein and a Parkinson's disease gene, is a sensor of lysosome stress or damage. Following lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires a signal at the damaged lysosome surface that releases an inhibited state of VPS13C, which hinders access of its VAB domain to lysosome-bound Rab7. Although another Parkinson's disease protein, LRRK2, is also recruited to stressed or damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the role of VPS13 proteins in bulk lipid transport, these findings suggest that lipid delivery to lysosomes by VPS13C is part of an early protective response to lysosome damage. Show less

Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinson's disease (PD). Here we show that VPS13C, a bridge-like lipid transport protein and a PD gene, is a sensor of lysosome stress/damage. Upon lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires a signal at the damaged lysosome surface that releases an inhibited state of VPS13C which hinders access of its VAB domain to lysosome-bound Rab7. While another PD protein, LRRK2, is also recruited to stressed/damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the role of VPS13 proteins in bulk lipid transport, these findings suggest that lipid delivery to lysosomes by VPS13C is part of an early protective response to lysosome damage. Show less

Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell-derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients' lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms. Show less

The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.60 and 2.97 Å resolution that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK, in cells exposed to submicromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular, and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggests that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases, including IRAK1, PIK3C3, PIP4K2C, and PIP5K3. Show less

The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location. Show less