Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

ABSTRACT: LRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective and closely related PPM1J/M exhibit virtually no activity toward substrates such as Rab8a phosphorylated at T72 (pT72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a conserved catalytic domain that adopts a β-sandwich fold. The striking difference is that PPM1H has evolved a 110-residue flap domain that punctuates the catalytic domain. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimera with the PPM1H flap domain dephosphorylates pT72 of Rab8a with a higher specific activity than PPM1H. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Parkinson's Disease

SUBMITTER: Yuko Pui Yiu Lam

LAB HEAD: Dario R. Alessi

PROVIDER: PXD026367 | Pride | 2021-08-11

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	20210426_YL_S10_PPM_D288A_T_SCX_14uL.raw	Raw
	20210426_YL_S11_PPM_D288A_T_A_3uL.raw	Raw
	20210426_YL_S12_PPM_D288A_T_G_3uL.raw	Raw
	20210426_YL_S13_PPM_WT_DSBU_T_5uL.raw	Raw
	20210426_YL_S15_PPM_WT__DSBU_T_A_5uL.raw	Raw

Items per page:

1 - 5 of 52

Similar Datasets

Development of a multiplex targeted mass spectrometry assay to quantify levels of LRRK2 phosphorylated Rab substrates and Ser910 and Ser935 biomarker sites

Project description:Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplex 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 phosphorylation of the LRRK2 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, lung, kidney and spleen). We define how each of the different pRab proteins are impacted by Parkinson’s pathogenic LRRK2[R1441C] and VPS35[D620N] mutations as well as 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 LRRK2 can phosphorylate Rab1 physiologically. We argue that this targeted mass spectrometry assay can replace immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

2021-03-17 | PXD022662 | Pride

Chlamydophila psittaci negative ocular adnexal marginal zone lymphomas express self polyreactive B cell receptors

Project description:The pathogenesis of Chlamydophila (C.) psittaci negative ocular adnexal extranodal marginal zone lymphomas (OAEMZLs) is poorly understood. OAEMZLs are monoclonal tumors expressing a biased repertoire of mutated surface immunoglobulins. Antigenic activation of the B cell receptor (BCR) may play a role in the pathogenesis of these lymphomas. We have analyzed the reactivity of recombinant OAEMZL immunoglobulins. OAEMZL antibodies reacted with self-human antigens, as demonstrated by enzyme-linked immunosorbent assays, HEp-2 immunofluorescence and human protein microarrays. All the analyzed recombinant antibodies (rAbs) exhibited polyreactivity by comprehensive protein array antibody reactivity and some rAbs also demonstrated rheumatoid factor activity. The identity of several reactive antigens was confirmed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry. The tested rAbs frequently reacted with shared intracellular and extracellular self-antigens (e.g. galectin-3). Furthermore, these self-antigens induced BCR signaling in B cells expressing cognate surface immunoglobulins derived from OAEMZLs. These findings suggest that interactions between self-antigens and cognate OAEMZL tumor-derived BCRs are functional, inducing intracellular signaling. Overall our findings suggest that self-antigen-induced BCR stimulation may be implicated in the pathogenesis of C. psittaci negative OAEMZLs. Antibody Specificity Profiling with four OAEMZL rAbs (Ab4438, Ab4726, Ab5334, and Ab11274) performed on ProtoArray Human Protein Microarrays

2015-01-30 | E-GEOD-65443 | biostudies-arrayexpress

Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases

Project description:Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson’s disease (PD). G2019S, the most common amino acid substitution activates the kinase two to three-fold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.

2016-02-04 | PXD003071 | Pride

4 RABs proxymity labelling - APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting

Project description:APEX2-mediated proximity labeling was performed with 4 endosomal RABs allowed an efficient and comprehensive identification of their protein neighbors. APEX2-mediated proximity labeling was performed in triplicate. Protein enrichments obtained from all RABs were compared to APEX2-nude proximity labeling results. Finally, bioinformatic and biochemical approaches were used to confirm functional validity of RAB protein neighbor’s identification.

2019-01-07 | PXD010950 | Pride

In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase

Project description:In higher plants, a P-type proton pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved but direct chemical evidence for which ones are specifically interacting with the C-terminus is lacking. In this study we used in vivo cross-linking experiments with a photo-reactive unnatural amino acid, p-benzoyl-phenylalanine (BPA) and tandem mass spectrometry, to obtain direct evidence that the carboxyl terminal regulatory domain directly interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a ‘head-to-tail’ organization of ATPase monomers in which the carboxyl terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and is consistent with prior studies suggesting that within the membrane, the protein can be organized as homopolymers, including dimers, trimers and hexamers.

2018-09-24 | PXD010892 | Pride

Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally Relevant Mutations: Analysis by Cross‐Linking, MS/MS, and NMR

Project description:The endogenous cellular prion protein (PrPC) can misfold into the scrapie isoform (PrPSc) and cause fatal infectious diseases. Despite significant research on the prion protein, both its normal function and whether alterations to that function play a critical role in prion diseases remain unknown. The protein consists of a predominantly alpha-helical C-terminal domain and an unstructured N-terminal domain that can coordinate Cu2+. Previous studies using NMR and EPR have revealed a tertiary association between the N-terminal domain and the C-terminal domain that we have hypothesized to be critical to the protein’s normal function. Here we investigated and quantified the inter-domain interactions within three different prion variants (wild type recombinant mouse PrPC, mutant delta central region (ΔCR), and disease mutant (E199K) after chemical cross-linking with a newly designed MS-cleavable reagent 1-(4-((2,5-Dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-4-(2-(3-methyl-3H-diazirin-3-yl)ethyl)-1,4-diazabicyclo[2.2.2] octane-1,4-diium (APDC4), followed by nHPLC(RP) and tandem MS analysis.

2020-06-16 | PXD012427 | Pride

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

Project description:Bacteria have evolved effectors or toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins regroups multidomain proteins with a modular organization, that comprise a C-terminal toxin domain fused to a N-terminal domain that adapt to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii. Here, we show that the Rhs polymorphic toxin forms a complex with the T6SS spike protein VgrG and a cognate chaperone EagR. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs complex formation requires the VgrG C-terminal β-helix and the Rhs Nterminal prePAAR and PAAR domains. We then report the cryo-electron-microscopy structure of the Rhs-EagR complex, demonstrating that the Rhs central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs protein through aspartyl autoproteolysis. We propose a model for Rhs loading on the T6SS, transport and delivery into the target cell.

2021-11-30 | PXD028652 | Pride

Quantitative cross-linking/mass spectrometry analysis revealed unknown structure of complement iC3

Project description:We applied quantitative cross-linking/mass spectrometry (QCLMS) to interrogate the structure of iC3 (or C3(H2O)), the activated hydrolytic product of the abundant human complement protein C3. The slow but spontaneous and ubiquitous formation of iC3 from C3 initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. QCLMS revealed structural differences and similarities between iC3 and C3, as well as between iC3 and C3b that is a pivotal proteolytic cleavage product of C3 and is functionally similar to iC3. Considered in combination with the crystal structures of C3 and C3b, our data support a model wherein the thioester-containing domain of C3 swings to the other end of the molecule creating, in iC3, a stable C3b-like platform for binding the zymogen, factor B, or the regulator, factor H. The integration of available crystallographic and QCLMS data allowed the determination of a 3D model for iC3. The unique arrangement of domains in iC3, which retains the anaphylatoxin (ANA) domain (while ANA is excised when C3 is enzymatically activated to C3b), is consistent with observed differences in activation and regulation between iC3 and C3b.

2016-06-17 | PXD003486 | Pride

Transcription profiling of peripheral blood or primary finroblast cultures from choroideremia patients to determine if loss-of-function mutations in REP-1 affect intracellular vesicle transport in fibroblasts and monocytes of CHM patients

Project description:Choroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion. To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. expression data were collected from 6 CHM patients' monocytes and 4 CHM primary fibroblasts cultures, monocytes or FB from 5 normal age-matched subjects were used as a control

2010-02-21 | E-GEOD-17549 | biostudies-arrayexpress

Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism

Project description:Rho GTPases regulate actin dynamics and cell motility, and their hyperactivation drives cancer metastasis1. P-Rex (PI(3,4,5)P3-dependent Rac Exchanger) guanine nucleotide exchange factors (GEFs) are potent on-switches for Rho GTPase signalling and are frequently dysregulated in metastatic cancer2. P-Rex GEFs are autoinhibited under basal conditions and activated synergistically by binding to Gβγ and PI(3,4,5)P33. However, the molecular basis for P-Rex autoinhibition remains unknown. Here we utilise X-ray crystallography, cryo-EM and cross-linking mass spectrometry to determine the autoinhibited P-Rex1 structure. P-Rex1 forms a characteristic bipartite structure with its seven domains split into distinct N- and C-terminal modules. The two modules are connected by a previously unrecognised C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the catalytic surface of the Dbl homology (DH) domain is occluded by the compact arrangement of the PH and DEP1 domains. Structural analysis of the DH-PH-DEP1 array reveals that a remarkable conformational transition, centred on a 126° opening of a hinge helix reminiscent of calmodulin dynamics, leads to the release of DH domain autoinhibition. Furthermore, given the off-axis position of the Gβγ and PI(3,4,5)P3 binding sites, our data suggest that concurrent Gβγ and PI(3,4,5)P3 requires counter-rotation of the two halves of P-Rex1 by 90°, leading to the uncoupling of the PH domain from the four-helic¬¬al bundle, and release of the autoinhibited DH domain to drive Rho GTPase signalling.

2022-08-12 | PXD034327 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data