In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson’s disease DOI Creative Commons
Adriana Cortés, Toan K. Phung, Lorena de Mena

и другие.

Brain, Год журнала: 2024, Номер unknown

Опубликована: Дек. 20, 2024

Abstract Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson’s disease (L2PD) and idiopathic PD (iPD). However, pharmaco-dynamic readouts progression biomarkers clinical trials aiming modification are insufficient since no endogenous marker reflecting enhanced activity of the most common LRRK2 G2019S mutation has been reported yet in L2PD patients. Employing phospho-/proteomic analyses we assessed impact that activating mutations had peripheral blood mononuclear cells (PBMCs) from cohort Spain (n=174). The groups study encompassed patients (n=37), non-manifesting carriers G2019S, here, L2NMCs (n=27), R1441G (n=14), (n=11), iPD (n=40), healthy controls (n=45). We identified 207 differential proteins compared to (39 up/168 down) 67 (10 up/57 down). down-regulated affected endolysosomal pathway, proteostasis, mitochondria, e.g., ATIC, RAB9A, or LAMP1. At phospho-proteome level, observed increases phosphorylation levels pSer106 RAB12 carriers, which were validated by immunoblotting after 1 year follow-up (n=48). Freshly collected PBMCs 3 L2PD, iPD, 5 (n=10) showed strong diminishment in-vitro administration MLi-2 inhibitor. Using machine learning, an 18-feature phospho-/protein signature discriminating L2NMCs, with 96% accuracy correlated severity, i.e., UPDRS-III motor scoring. easily accessible cohort, elevated as biomarker carriers. Our data suggest monitoring could be relevant tracking activation, particularly Future work may determine whether help patient enrichment drug efficacy trials.

Язык: Английский

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system DOI Creative Commons
Olawale G. Raimi, Hina Ojha, Kenneth Ehses

и другие.

Science Advances, Год журнала: 2024, Номер 10(23)

Опубликована: Июнь 7, 2024

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson’s disease (PD). Stabilization PINK1 at the translocase outer membrane (TOM) complex damaged mitochondria is critical for its activation. The mechanism how activated TOM unclear. Here, we report that co-expression human and all seven subunits Saccharomyces cerevisiae sufficient We use this reconstitution system to systematically assess role each subunit toward unambiguously demonstrate TOM20 TOM70 receptor required optimal activation map their sites interaction with using AlphaFold structural modeling mutagenesis. also an essential pore-containing TOM40 structurally associated TOM7 TOM22 These findings will aid development small-molecule activators as therapeutic strategy PD.

Язык: Английский

Процитировано

15
CURTAIN—A unique web-based tool for exploration and sharing of MS-based proteomics data DOI Creative Commons
Toan K. Phung, Kerryn Berndsen, R. Shastry

и другие.

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(7)

Опубликована: Фев. 7, 2024

To facilitate analysis and sharing of mass spectrometry (MS)-based proteomics data, we created online tools called CURTAIN (https://curtain.proteo.info) CURTAIN-PTM (https://curtainptm.proteo.info) with an accompanying series video tutorials (https://www.youtube.com/@CURTAIN-me6hl). These are designed to enable non-MS experts interactively peruse volcano plots deconvolute primary experimental data so that replicates can be visualized in bar charts or violin exported publication-ready format. They also allow assessment overall quality by correlation matrix profile plot analysis. After making a selection protein "hits", the user analyze known domain structure, AlphaFold predicted reported interactors, relative expression as well disease links. permits all identified PTM sites on protein(s) interest selected databases. links Kinase Library predict upstream kinases may phosphorylate interest. We provide examples utility analyzing how targeted degradation PPM1H Rab phosphatase counteracts Parkinson's LRRK2 kinase impacts cellular levels phosphorylation sites. reanalyzed ubiquitylation dataset, characterizing PINK1-Parkin pathway activation neurons, revealing not highlighted previously. free use open source, enabling researchers share maximize impact their data. advocate MS published format containing shareable weblink, thereby allowing readers better exploit

Язык: Английский

Процитировано

9
Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA DOI Creative Commons

Abigail Brewer,

Gajanan Sathe,

Billie E. Pflug

и другие.

iScience, Год журнала: 2024, Номер 27(3), С. 109302 - 109302

Опубликована: Фев. 19, 2024

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal subunit. full scope of substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras efficiently selectively degrade dTAG-PPP2CA homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins significantly increased phosphorylation upon degradation, implicating them as potential substrates. A vast majority these are novel. Bioinformatic analyses revealed involvement spliceosome function, cell cycle, RNA transport, ubiquitin-mediated proteolysis. We identify pSP/pTP motif predominant target for confirm some our phospho-proteomic data immunoblotting. provide in-depth atlas establish targeted degradation robust tool unveil

Язык: Английский

Процитировано

8
Parkinson’s VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B DOI Creative Commons
Prosenjit Pal, Matthew Taylor, Yuko P. Y. Lam

и другие.

Science Advances, Год журнала: 2023, Номер 9(50)

Опубликована: Дек. 13, 2023

We demonstrate that the Parkinson's VPS35[D620N] mutation alters expression of ~220 lysosomal proteins and stimulates recruitment phosphorylation Rab at lysosome. This recruits phospho-Rab effector protein RILPL1 to lysosome where it binds integral membrane TMEM55B. identify highly conserved regions TMEM55B interact design mutations block binding. In mouse fibroblasts, brain, lung, we reduces levels, in a manner reversed by LRRK2 inhibition proteasome inhibitors. Knockout enhances substrates, knockout increases levels. The lysosomotropic agent LLOMe also induced kinase-mediated association lysosome, but lower extent than D620N mutation. Our study uncovers pathway through which dysfunctional lysosomes resulting from recruit activate on surface, driving assembly RILPL1-TMEM55B complex.

Язык: Английский

Процитировано

16
Tagless LysoIP method for molecular profiling of lysosomal content in clinical samples DOI Creative Commons
Daniel Saarela, Paweł Lis, Sara Gomes

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Май 19, 2024

Abstract Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling content using tag-based immunoprecipitation (LysoTagIP) cell animal models allowed major discoveries the field, however studying dysfunction patients remains challenging. Here, we report development “tagless LysoIP method” to enable rapid enrichment lysosomes, via immunoprecipitation, endogenous integral membrane protein TMEM192, directly from clinical samples lines (e.g. induced Pluripotent Stem Cell (iPSCs) derived neurons). Isolated lysosomes intact suitable for subsequent multimodal omics analyses. To validate our approach, employed tagless enrich peripheral blood mononuclear cells (PBMCs) fresh with CLN3 disease, neurodegenerative LSD. Metabolic profiling isolated showed massive accumulation glycerophosphodiesters (GPDs) patients’ lysosomes. Interestingly, patient milder phenotype genotype displayed lower GPDs, consistent their potential role as disease biomarkers. Altogether, provides framework study native samples, identify novel biomarkers discover human-relevant mechanisms.

Язык: Английский

Процитировано

5
Ubiquitylomics: An Emerging Approach for Profiling Protein Ubiquitylation in Skeletal Muscle DOI Creative Commons
Samuel O. Lord, Harvey E. Johnston, Rahul S. Samant

и другие.

Journal of Cachexia Sarcopenia and Muscle, Год журнала: 2024, Номер unknown

Опубликована: Сен. 16, 2024

ABSTRACT Skeletal muscle is a highly adaptable tissue, finely tuned by various physiological and pathological factors. Whilst the pivotal role of skeletal in overall health widely acknowledged, unravelling underlying molecular mechanisms poses ongoing challenges. Protein ubiquitylation, crucial post‐translational modification, involved regulating most biological processes. This widespread impact achieved through diverse set enzymes capable generating structurally functionally distinct ubiquitin modifications on proteins. The complexity protein ubiquitylation has presented significant challenges not only identifying ubiquitylated proteins but also characterising their functional significance. Mass spectrometry enables in‐depth analysis modification status, offering powerful tool for studying its diversity: an approach termed ubiquitylomics. Ubiquitylomics been employed to tackle different perspectives including limited global quantification substrates linkages, site recognition crosstalk with other modifications. As field mass continues evolve, usage ubiquitylomics unravelled novel insights into regulatory governing biology. However, research predominantly conducted cellular models, limiting our understanding signalling events driving By integrating intricate landscape dynamic shifts physiology, promises deepen biology lay foundation developing transformative muscle‐related therapeutics. review aims articulate how can be utilised researchers address aspects muscle. We explore methods used experiments, highlight relevant literature employing context outline considerations experimental design.

Язык: Английский

Процитировано

1
NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress DOI Creative Commons

Ramchandra V. Amnekar,

Toby A. Dite, Paweł Lis

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Дек. 17, 2024

Abstract WNK family kinases are regulated by osmotic stress and control ion homeostasis activating SPAK OXSR1 kinases. Using a proximity ligation approach, we found that promotes the association of WNK1 with NRBP1 pseudokinase TSC22D2/4 adaptor proteins. is closely related to isoforms also contains RΦ-motif binding conserved C-terminal (CCT) domain, similar CCT domains in WNKs, OXSR1. Knockdown markedly inhibited sorbitol-induced activation downstream components. AlphaFold-3 modelling predicts WNK1, SPAK, NRBP1, TSC22D4 form complex, which two RΦ-motifs interact CCTL1 domain NRBP1. Our data indicate functions as an upstream activator pathway.

Язык: Английский

Процитировано

1
In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson DOI Creative Commons
Adriana Cortés, Toan K. Phung, Lorena de Mena

и другие.

medRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Май 6, 2024

ABSTRACT Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson’s disease (L2PD) and idiopathic PD (iPD). Yet, pharmaco-dynamic readouts progression biomarkers modification clinical trials are insufficient. Employing phospho-/proteomic analyses we assessed the impact that LRRK2 activating mutations had in peripheral blood mononuclear cells (PBMCs) from cohort Spain (n=174) encompassing G2019S L2PD patients (n=37), non-manifesting mutation carriers of G2019S, here, L2NMCs (n=27), R1441G (n=14), (n=11), iPD (n=40), controls (n=45). We identified 207 differential proteins compared to (39 up/ 168 down) 67 (10 57 down). down-regulated affected endolysosomal pathway, proteostasis mitochondria, e.g., ATIC, RAB9A, or LAMP1. At phospho-proteome level, observed increases endogenous phosphorylation levels pSer106 RAB12 carriers, which were validated by immunoblotting after 1 year follow-up (n=48). Freshly collected PBMCs 3 L2PD, iPD, 5 (n=10) showed strong diminishment in-vitro administration MLi-2 inhibitor. Using machine learning, an 18-feature phospho-/protein signature capable discriminating L2NMCs, with 96% accuracy correlated severity, i.e., UPDRS-III motor scoring. Our study as biomarker easily accessible suggests findings human such can be deployed universal readout iPD. Future work may determine whether could help patient enrichment monitoring drug efficacy trials.

Язык: Английский

Процитировано

0
In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson’s disease DOI Creative Commons
Adriana Cortés, Toan K. Phung, Lorena de Mena

и другие.

Brain, Год журнала: 2024, Номер unknown

Опубликована: Дек. 20, 2024

Abstract Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson’s disease (L2PD) and idiopathic PD (iPD). However, pharmaco-dynamic readouts progression biomarkers clinical trials aiming modification are insufficient since no endogenous marker reflecting enhanced activity of the most common LRRK2 G2019S mutation has been reported yet in L2PD patients. Employing phospho-/proteomic analyses we assessed impact that activating mutations had peripheral blood mononuclear cells (PBMCs) from cohort Spain (n=174). The groups study encompassed patients (n=37), non-manifesting carriers G2019S, here, L2NMCs (n=27), R1441G (n=14), (n=11), iPD (n=40), healthy controls (n=45). We identified 207 differential proteins compared to (39 up/168 down) 67 (10 up/57 down). down-regulated affected endolysosomal pathway, proteostasis, mitochondria, e.g., ATIC, RAB9A, or LAMP1. At phospho-proteome level, observed increases phosphorylation levels pSer106 RAB12 carriers, which were validated by immunoblotting after 1 year follow-up (n=48). Freshly collected PBMCs 3 L2PD, iPD, 5 (n=10) showed strong diminishment in-vitro administration MLi-2 inhibitor. Using machine learning, an 18-feature phospho-/protein signature discriminating L2NMCs, with 96% accuracy correlated severity, i.e., UPDRS-III motor scoring. easily accessible cohort, elevated as biomarker carriers. Our data suggest monitoring could be relevant tracking activation, particularly Future work may determine whether help patient enrichment drug efficacy trials.

Язык: Английский

Процитировано

0