Biology subjects

Lis, P.

Publications and source records attributed to Lis, P..

12 recordsLinked to original sources

bioRxiv2025

Alternate Splicing Directs PMCA2 to Lysosomes and is Linked to Neurodegeneration

Plasma membrane calcium ATPases (PMCAs) are believed to function exclusively at the plasma membrane where they expel calcium from the cytosol. We have unexpectedly identified a splice variant-dependent localisation of the PMCA isoform PMCA2 to the lysosome, where it forms an evolutionarily conserved complex with NPC1, the lysosomal membrane protein defective in the rare lysosomal storage disease Niemann-Pick disease type C (NPC). This interaction is required for lysosomal Ca2+ homeostasis and implicates PMCA2 as a mediator of Ca2+ uptake into lysosomes. Disruption of the NPC1-PMCA2 complex contributes to the pathophysiology of both Niemann-Pick disease type C and Parkinsons disease, revealing an unrecognised intracellular function for PMCA2 and a shared mechanism linking lysosomal Ca2+ and lipid regulation in neurodegeneration.

cell biology↗

bioRxiv2024

NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress

WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity ligation approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation and mass spectrometry and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a R{Phi}-motif binding conserved C-terminal (CCT) domain, similar to the CCT domains in WNKs, SPAK and OXSR1. Knockdown or knock-out of NRBP1 markedly inhibited sorbitol-induced activation of WNK1 and downstream components. We demonstrate recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modelling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 R{Phi}-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicates NRBP1 functions as an upstream activator of the WNK pathway. TeaserNRBP1 functions as a scaffolding component regulating the assembly of a multi-subunit complex, required for the activation of the WNK Lysine Deficient Protein Kinase family in response to osmotic stress. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/628181v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5b37eborg.highwire.dtl.DTLVardef@b4661corg.highwire.dtl.DTLVardef@19bc3e9org.highwire.dtl.DTLVardef@15b052e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Tagless LysoIP method for molecular profiling of lysosomal content in clinical samples

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 allowed major discoveries in the field, however studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the "tagless LysoIP method" to enable 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 (iPSCs) derived neurons). Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood from patients with CLN3 disease, a neurodegenerative LSD. Metabolic profiling of isolated lysosomes showed 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 novel biomarkers and discover human-relevant disease mechanisms.

cell biology↗

bioRxiv2023

Pulses of RhoA Signaling Stimulate Actin Polymerization and Flow in Protrusions to Drive Collective Cell Migration

In animals, cells often move as collectives to shape organs, close wounds, or--in the case of disease--metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network (Yamada and Sixt, 2019). Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin II-dependent actin flow and protrusion retraction at the base of the protrusions, and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other--but not all (Bastock and Strutt, 2007; Lebreton and Casanova, 2013; Matthews et al., 2008)--contexts.

developmental biology↗

bioRxiv2023

Parkinson's VPS35 mutation induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B

The Parkinsons VPS35[D620N] mutation causes lysosome dysfunction enhancing LRRK2 kinase activity. We find the VPS35[D620N] mutation alters expression of [~]350 lysosomal proteins and stimulates LRRK2 phosphorylation of Rab proteins at the lysosome. This recruits the phosphoRab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35 [D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition. Knock-out of RILPL1 enhances phosphorylation of Rab substrates and knock-out of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe, also induced LRRK2 kinase mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.

neuroscience↗

bioRxiv2022

Interaction with C21ORF2 controls the cellular functions of the NEK1 kinase

NEK1 is a pleiotropic protein kinase implicated in mitosis, ciliogenesis and DNA repair but little is known about its regulation or targets. Its relevance for human health is underscored by the association of NEK1 mutations with human diseases including axial spondylometaphyseal dyplasia (SMD) and amyotrophic lateral sclerosis (ALS). Mutations in the C21ORF2 gene are associated with a similar pattern of human diseases, suggesting close functional links with NEK1. Here we report that in unperturbed, untransformed cells, endogenous NEK1 and C21ORF2 form a tight complex that does not appear to contain other proteins. A small acidic domain "CID: C21ORF2 interaction domain" at the C-terminus of NEK1 is necessary and sufficient to interact with C21ORF2, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts with high confidence an extended binding interface between a leucine-rich repeat (LRR) domain in the N-terminal half of C21ORF2 and a stretch of the NEK1-CID; mutating residues mediating electrostatic interactions within this interface disrupts the NEK1-C21ORF2 interaction. This model also explains why pathogenic mutations disrupt the complex. We go on to show that the kinase activity of NEK1 and its interaction with C21ORF2 is critical for NEK1 function in cells. These data reveal C21ORF2 as a regulatory subunit of NEK1, illuminating our understanding of how this kinase is regulated and NEK1-C21ORF2-associated diseases.

biochemistry↗

bioRxiv2022

Endogenous Rab38 regulates LRRK2's membrane recruitment and substrate Rab phosphorylation in melanocytes

Point mutations in LRRK2 cause Parkinsons Disease and augment LRRK2s kinase activity. However, cellular pathways that enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here we identify Rab38 as a novel physiological regulator of LRRK2. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown of Rab38 but not Rab32 or Rab29 decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both exogenous and endogenous LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association, and overexpressed kinase-active but not kinase-inactive LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Deletion or mutation of LRRK2 at the Rab38 binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. Consistently, overexpression of LRRK2350-550, a fragment that encompasses the Rab38 binding site, blocks endogenous LRRK2s phosphorylation of Thr73-Rab10. Finally, disruption of BLOC-3, the guanine nucleotide exchange factor for Rab38 and 32, inhibits Rab38s regulation of LRRK2. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.

cell biology↗

bioRxiv2022

A Feed-forward Pathway Drives LRRK2 kinaseMembrane Recruitment and Apparent Activation

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinsons disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed "Site #1", can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed "Site #2", that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

biochemistry↗

bioRxiv2020

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinsons disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinsons mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

biochemistry↗

bioRxiv2020

Development of a multiplexed targeted mass spectrometry assay for LRRK2 phosphorylated Rabs and Ser910/Ser935 biomarker sites

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinsons pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinsons patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

neuroscience↗

bioRxiv2020

Rab29 knock-out or transgenic overexpression does not impact basal LRRK2 activity in wildtype and pathogenic mouse and cell line models

Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinsons disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinsons, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans Golgi network. Here we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on assessment of Rab10 and Rab12 phosphorylation, in wildtype LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that monovalent cation ionophore antibiotics nigericin and monensin enhance LRRK2-mediated Rab10 and Rab12 phosphorylation 4 to 9-fold, in a manner that is independent from Rab29. Moderate stimulation of Rab10 and Rab12 induced by lysosome stressors chloroquine and LLOMe was also not regulated by Rab29. Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes.

neuroscience↗

bioRxiv2019

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

Mutations that activate LRRK2 protein kinase cause Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knock out increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A \"substrate-trapping\" PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinsons disease.

biochemistry↗