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Missense mutations in park2, encoding the parkin protein, account for ϳ50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ϳ160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys 150 -Cys 169 and Cys 196 -His 215 consisting of CX 2 -3 CX 11 CX 2 C and CX 4 -6 CX 10 -16 -CX 2 (H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn 2؉ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease. Autosomal recessive juvenile Parkinson disease (ARJP)2 is a neurodegenerative disorder arising from the loss of dopaminergic neurons in the substantia nigra of the midbrain. ARJP is characterized by the onset of Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is distinguished from the idiopathic form of Parkinson disease by the onset of symptoms, prior to the age of forty. The hereditary nature of ARJP implicates a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2, and DJ-1 as the cause of dopaminergic neurodegeneration (1-4). A variety of deletion, truncation, and point mutations distributed throughout the park2 gene, which encodes the protein parkin, have been reported in ARJP patients (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR (RING-between-RING) ubiquitin li...

Introduction: Cancer is often diagnosed at late stages when the chance of cure is relatively low and although research initiatives in oncology discover many potential cancer biomarkers, few transition to clinical applications. This review addresses the current landscape of cancer biomarker discovery and translation with a focus on proteomics and beyond. Areas covered: The review examines proteomic and genomic techniques for cancer biomarker detection and outlines advantages and challenges of integrating multiple omics approaches to achieve optimal sensitivity and address tumor heterogeneity. This discussion is based on a systematic literature review and direct participation in translational studies. Expert commentary: Identifying aggressive cancers early on requires improved sensitivity and implementation of biomarkers representative of tumor heterogeneity. During the last decade of genomic and proteomic research, significant advancements have been made in next generation sequencing and mass spectrometry techniques. This in turn has led to a dramatic increase in identification of potential genomic and proteomic cancer biomarkers. However, limited successes have been shown with translation of these discoveries into clinical practice. We believe that the integration of these omics approaches is the most promising molecular tool for comprehensive cancer evaluation, early detection and transition to Precision Medicine in oncology.

Mutations in Parkin are one of the predominant hereditary factors found in patients suffering from autosomal recessive juvenile Parkinsonism. Parkin is a member of the E3 ubiquitin ligase family that is defined by a tripartite RING1-in-between-ring (IBR)-RING2 motif. In Parkin, the IBR domain has been shown to augment binding of the E2 proteins UbcH7 and UbcH8, and the subsequent ubiquitination of the proteins synphilin-1, Sept5, and SIM2. To facilitate our understanding of Parkin function, the solution structure of the Parkin IBR domain was solved by using NMR spectroscopy. Folding of the IBR domain (residues M327-S378) was found to be zinc dependent, and the structure reveals the domain forms a unique pair scissor-like and GAG knuckle-like zinc-binding sites, different from other zinc-binding motifs such as the RING, LIM, PHD, or B-box motifs. The N terminus of the IBR domain, residues E307-E322, is unstructured. The disease causing mutation T351P causes global unfolding, whereas the mutation R334C causes some structural rearrangement of the domain. In contrast, the protein containing the mutation G328E appears to be properly folded. The structure of the Parkin IBR domain, in combination with mutational data, allows a model to be proposed where the IBR domain facilitates a close arrangement of the adjacent RING1 and RING2 domains to facilitate protein interactions and subsequent ubiquitination.ubiquitination ͉ zinc-binding ͉ NMR spectroscopy ͉ protein folding ͉ protein interactions P arkinson's disease (PD) is a common neurodegenerative disease characterized by damage to the dopaminergic neurons of the substantia nigra of the midbrain manifesting itself with symptoms such as tremors, rigidity, and bradykinesia (1, 2). Autosomal recessive juvenile PD (ARJP) is an early-onset (Ͻ40 years of age) form of the disease that is caused by hereditary factors including mutations in the genes DJ-1, PINK1, and in about half of the cases, parkin. The protein Parkin has been identified as an E3 ubiquitin-protein ligase of the ubiquitin-proteosome system that is required to maintain cellular protein quality control by removing misfolded or damaged proteins (3, 4). Ubiquitin mediated proteolysis occurs through a cascade of ubiquitin transfers from an E1 activating enzyme, to an E2 conjugating enzyme and finally in complex with an E3 ligase to the target substrate (5, 6). At least 40 different missense and deletion mutations in the parkin gene have been identified and correlated to dysfunction of the ubiquitination system, manifesting itself as ARJP due to the loss of the dopaminergic neurons (7).Parkin is a 465-residue protein comprising an N-terminal ubiquitin-like domain, a unique Parkin-specific domain in the central region, and two RING finger domains (RING1, RING2) separated by an in-between ring (IBR) or double ring linked domain near its C terminus. The cysteine and histidine rich RING-IBR-RING (RBR) domain architecture is highly conserved and found only in eukaryotes. To date, all characterized proteins containing the ...

Phosphorylation of MDM2 by ATM upon DNA damage is an important mechanism for deregulating MDM2, thereby leading to p53 activation. ATM phosphorylates multiple residues near the RING domain of MDM2, but the underlying molecular basis for deregulation remains elusive. Here we show that Ser429 phosphorylation selectively enhances the ubiquitin ligase activity of MDM2 homodimer but not MDM2-MDMX heterodimer. A crystal structure of phospho-Ser429 (pS429)-MDM2 bound to E2-ubiquitin reveals a unique 3 10helical feature present in MDM2 homodimer that allows pS429 to stabilize the closed E2-ubiquitin conformation and thereby enhancing ubiquitin transfer. In cells Ser429 phosphorylation increases MDM2 autoubiquitination and degradation upon DNA damage, whereas S429A substitution protects MDM2 from auto-degradation. Our results demonstrate that Ser429 phosphorylation serves as a switch to boost the activity of MDM2 homodimer and promote its self-destruction to enable rapid p53 stabilization and resolve a long-standing controversy surrounding MDM2 auto-degradation in response to DNA damage.

Background:The RING E3 AO7/RNF25 binds its E2 with unusually high affinity. Results: AO7 has a secondary E2 binding site adjacent to the RING. Conclusion: This site prevents the stimulatory effect of non-covalent backside binding of ubiquitin, has unique agonist properties, and allows for structural analysis of RING mutants. Significance: Knowledge of how RING E3s mediate ubiquitination is critical to understanding cellular protein regulation.

Combining human genomics with proteomics is becoming a powerful tool for drug discovery. Associations between genetic variants and protein levels can uncover disease mechanisms, clinical biomarkers, and candidate drug targets. To date, most population-level proteogenomic studies have focused on common alleles through genome-wide association studies (GWAS). Here, we studied the contribution of rare protein-coding variants to 1,472 plasma proteins abundances measured via the Olink Explore 1536 assay in 50,829 UK Biobank human exomes. Through a variant-level exome-wide association study (ExWAS), we identified 3,674 rare and significant protein quantitative trait loci (pQTLs), of which 76% were undetected in a prior GWAS performed on the same cohort, and we found that rare pQTLs are less likely to be random in their variant effect annotation. In gene-based collapsing analyses, we identified an additional 166 significant gene-protein pQTL signals that were undetected through single-variant analyses. Of the total 456 protein-truncating variant (PTV)-driven cis-pQTLs in the gene-based collapsing analysis, 99.3% were associated with decreased protein levels. We demonstrate how this resource can identify allelic series and propose biomarkers for several candidate therapeutic targets, including GRN, HSD17B13, NLRC4, and others. Finally, we introduce a new collapsing analysis framework that combines PTVs with missense cis-pQTLs that are associated with decreased protein abundance to bolster genetic discovery statistical power. Our results collectively highlight a considerable role for rare variation in plasma protein abundance and demonstrate the utility of plasma proteomics in gene discovery and unravelling mechanisms of action.

RationalePulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and anti-pathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD.ObjectivesWe investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights about potential disease mechanisms.MethodsBronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed.ResultsTotal phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and surfactant protein (SP)-B, SP-A and SP-D concentrations were lower, COPDversuscontrols, log2 fold change (log2FC)=−2.0, −2.2, −1.5, −0.5, −0.7, −0.5 (adj. p-value<0.02), respectively, and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and SP-A, SP-B, SP-D, NAPSA and CD44 inversely correlated with CT small airways disease measures (E/I MLD), r=−0.56, r=−0.58, r=−0.45, r=−0.36, r=−0.44, r=−0.37, r=−0.40, r=−0.39 (adj. p-value<0.05). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (%LAA): r=−0.55, r=−0.61, r=−0.48, r=−0.51, r=−0.41, r=−0.31, r=−0.34, respectively (adj. p-value<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated, COPDversuscontrols, log2FC of 0.40 (adj. p-value=0.0390) and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPDversusHV-ES, and predicted COPD status, AUC=0.85.ConclusionsUsing a multiomics approach we, for the first time, demonstrate global surfactant dysregulation in COPD which was associated with emphysema giving new insights about potential mechanisms underlying the cause or consequence of disease.