ABSTRACT
The therapeutic effects of l-3,4-dihydroxyphenylalanine (l-DOPA) in patients with Parkinson's disease (PD) severely diminishes with the onset of abnormal involuntary movement, l-DOPA-induced dyskinesia (LID). However, the molecular mechanisms that promote LID remain unclear. Here, we demonstrated that RasGRP1 [(guanine nucleotide exchange factor (GEF)] controls the development of LID. l-DOPA treatment rapidly up-regulated RasGRP1 in the striatum of mouse and macaque model of PD. The lack of RasGRP1 in mice (RasGRP1-/- ) dramatically diminished LID without interfering with the therapeutic effects of l-DOPA. Besides acting as a GEF for Ras homolog enriched in the brain (Rheb), the activator of the mammalian target of rapamycin kinase (mTOR), RasGRP1 promotes l-DOPA-induced extracellular signal-regulated kinase (ERK) and the mTOR signaling in the striatum. High-resolution tandem mass spectrometry analysis revealed multiple RasGRP1 downstream targets linked to LID vulnerability. Collectively, the study demonstrated that RasGRP1 is a critical striatal regulator of LID.
Subject(s)
Dyskinesia, Drug-Induced , Parkinson Disease , Animals , Corpus Striatum , DNA-Binding Proteins , Disease Models, Animal , Dyskinesia, Drug-Induced/etiology , Guanine Nucleotide Exchange Factors/genetics , Humans , Levodopa/adverse effects , Mammals , Parkinson Disease/etiology , Parkinson Disease/genetics , TOR Serine-Threonine KinasesABSTRACT
The striatum of the brain coordinates motor function. Dopamine-related drugs may be therapeutic to patients with striatal neurodegeneration, such as Huntington's disease (HD) and Parkinson's disease (PD), but these drugs have unwanted side effects. In addition to stimulating the release of norepinephrine, amphetamines, which are used for narcolepsy and attention-deficit/hyperactivity disorder (ADHD), trigger dopamine release in the striatum. The guanosine triphosphatase Ras homolog enriched in the striatum (Rhes) inhibits dopaminergic signaling in the striatum, is implicated in HD and L-dopa-induced dyskinesia, and has a role in striatal motor control. We found that the guanine nucleotide exchange factor RasGRP1 inhibited Rhes-mediated control of striatal motor activity in mice. RasGRP1 stabilized Rhes, increasing its synaptic accumulation in the striatum. Whereas partially Rhes-deficient (Rhes+/-) mice had an enhanced locomotor response to amphetamine, this phenotype was attenuated by coincident depletion of RasGRP1. By proteomic analysis of striatal lysates from Rhes-heterozygous mice with wild-type or partial or complete knockout of Rasgrp1, we identified a diverse set of Rhes-interacting proteins, the "Rhesactome," and determined that RasGRP1 affected the composition of the amphetamine-induced Rhesactome, which included PDE2A (phosphodiesterase 2A; a protein associated with major depressive disorder), LRRC7 (leucine-rich repeat-containing 7; a protein associated with bipolar disorder and ADHD), and DLG2 (discs large homolog 2; a protein associated with chronic pain). Thus, this Rhes network provides insight into striatal effects of amphetamine and may aid the development of strategies to treat various neurological and psychological disorders associated with the striatal dysfunction.
Subject(s)
Amphetamine/pharmacology , Behavior, Animal/drug effects , GTP-Binding Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Locomotion/drug effects , Signal Transduction/drug effects , Animals , GTP-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , HEK293 Cells , Humans , Mice, Mutant Strains , Rats , Signal Transduction/geneticsABSTRACT
Liver receptor homologue-1 (LRH1) is an orphan nuclear receptor that has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the LRH1 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative LRH1 co-regulators. Nuclear fractionation protocol was essential for detection of LRH1 peptides by mass spectrometry (MS), with most peptides being observed in the insoluble fraction (receptor bound to DNA). SERBP1 and ILF3 were identified as LRH1 interacting partners by both Western blot and MS/MS analysis. Receptor knockdown by siRNA showed an increase in SERBP1 expression, while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of LRH1 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, subsequently inhibiting transcription. Given the receptor's role in cancer progression, the study here elucidates additional transcriptional machinery involved in LRH1 signaling and potentially provides new targets for therapeutics development.
Subject(s)
Gene Expression Regulation , Peptides/analysis , RNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription, Genetic , Amino Acid Sequence , Cell Line, Tumor , Cell Nucleus/metabolism , Chemical Fractionation , HEK293 Cells , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Molecular Sequence Annotation , Molecular Sequence Data , Nuclear Factor 90 Proteins/genetics , Nuclear Factor 90 Proteins/metabolism , Peptides/genetics , Peptides/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Plasmids/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Proteolysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , TransfectionABSTRACT
The goal in proteomics to identify all peptides in a complex mixture has been largely addressed using various LC MS/MS approaches, such as data dependent acquisition, SRM/MRM, and data independent acquisition instrumentation. Despite these developments, many peptides remain unsequenced, often due to low abundance, poor fragmentation patterns, or data analysis difficulties. Many of the unidentified peptides exhibit strong evidence in high resolution MS(1) data and are frequently post-translationally modified, playing a significant role in biological processes. Proteomics Workbench (PWB) software was developed to automate the detection and visualization of all possible peptides in MS(1) data, reveal candidate peptides not initially identified, and build inclusion lists for subsequent MS(2) analysis to uncover new identifications. We used this software on existing data on the autophagy regulating kinase Ulk1 as a proof of concept for this method, as we had already manually identified a number of phosphorylation sites Dorsey, F. C. et al (J. Proteome. Res. 8(11), 5253-5263 (2009)). PWB found all previously identified sites of phosphorylation. The software has been made freely available at http://www.proteomicsworkbench.com . Graphical Abstract á .
