Parkin function in Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disease, and is characterized by involuntary shaking, muscle rigidity, and the progressive loss of dopaminergic neurons. In ∼5 to 10% of PD cases there is a genetic association, with almost 20 genes attributed to date. One example is early-onset autosomal recessive PD (ARPD), for which the majority of cases are linked to mutations in the Parkin gene (PRKN; also known as PARK2). PRKN encodes the E3 ubiquitin ligase Parkin, which plays important roles in mitochondrial quality control and turnover. Parkin, although localized to the mitochondria under certain conditions, is primarily cytosolic (1). A second ARPD-associated gene, PINK1 (PTEN-induced putative kinase 1), encodes a mitochondrially tethered kinase that regulates Parkin activity through phosphorylation events. Mutations in PINK1, although rare, are associated with a phenotype similar to that of ARPD patients with PRKN mutations. Numerous mutations throughout PRKN are linked to ARPD, making the functional examination of Parkin crucial to understanding ARPD pathogenesis. A wealth of structural studies have transformed our knowledge of Parkin regulation and catalytic mechanisms. However, the current picture is incomplete, leading to several possible models of Parkin catalysis, which has implications for understanding how the ARPD-associated mutations affect the protein and thus PD pathogenesis

Selenium incorporation using recombinant techniques

An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein

RBR E3 ubiquitin ligases: new structures, new insights, new questions

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. INTRODUCTIO

Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ~40 E2s and ~600 E3s giving rise to a possible ~24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL

Towards a Molecular Understanding of the Fanconi Anemia Core Complex

Fanconi Anemia (FA) is a genetic disorder characterized by the inability of patient cells to repair DNA damage caused by interstrand crosslinking agents. There are currently 14 verified FA genes, where mutation of any single gene prevents repair of DNA interstrand crosslinks (ICLs). The accumulation of ICL damage results in genome instability and patients having a high predisposition to cancers. The key event of the FA pathway is dependent on an eight-protein core complex (CC), required for the monoubiquitination of each member of the FANCD2-FANCI complex. Interestingly, the majority of patient mutations reside in the CC. The molecular mechanisms underlying the requirement for such a large complex to carry out a monoubiquitination event remain a mystery. This paper documents the extensive efforts of researchers so far to understand the molecular roles of the CC proteins with regard to its main function in the FA pathway, the monoubiquitination of FANCD2 and FANCI

Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1

The Fanconi anemia pathway for DNA interstrand crosslink repair and the translesion synthesis pathway for DNA damage tolerance both require cycles of monoubiquitination and deubiquitination. The ubiquitin-specific protease-1 (USP1), in complex with USP1-associated factor 1, regulates multiple DNA repair pathways by deubiquitinating monoubiquitinated Fanconi anemia group D2 protein (FANCD2), Fanconi anemia group I protein (FANCI), and proliferating cell nuclear antigen (PCNA). Loss of USP1 activity gives rise to chromosomal instability. Whereas many USPs hydrolyse ubiquitin–ubiquitin linkages, USP1 targets ubiquitin–substrate conjugates at specific sites. The molecular basis of USP1's specificity for multiple substrates is poorly understood. Here, we reconstitute deubiquitination of purified monoubiquitinated FANCD2, FANCI, and PCNA and show that molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. We found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. Furthermore, we show that the N-terminus of USP1 is sufficient to engineer specificity in a more promiscuous USP

Mind the metal:a fragment library-derived zinc impurity binds the E2 ubiquitin-conjugating enzyme Ube2T and induces structural rearrangements

Efforts to develop inhibitors, activators, and effectors of biological reactions using small molecule libraries are often hampered by interference compounds, artifacts, and false positives that permeate the pool of initial hits. Here, we report the discovery of a promising initial hit compound targeting the Fanconi anemia ubiquitin-conjugating enzyme Ube2T and describe its biophysical and biochemical characterization. Analysis of the co-crystal structure led to the identification of a contaminating zinc ion as solely responsible for the observed effects. Zinc binding to the active site cysteine induces a domain swap in Ube2T that leads to cyclic trimerization organized in an open-ended linear assembly. Our study serves as a cautionary tale for screening small molecule libraries and provides insights into the structural plasticity of ubiquitin-conjugating enzymes

Characterization of FANCL variants observed in patient cancer cells

Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand crosslinks. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognising the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand crosslinking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilising, with N270K and E289Q destabilising the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson’s disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin’s N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity

Allosteric targeting of the Fanconi anemia ubiquitin-conjugating enzyme Ube2T by fragment screening

Ube2T is the E2 ubiquitin-conjugating enzyme of the Fanconi anemia DNA repair pathway and it is overexpressed in several cancers, representing an attractive target for the development of inhibitors. Despite the extensive efforts in targeting the ubiquitin system, very few E2 binders have currently been discovered. Herein we report the identification of a new allosteric pocket on Ube2T through a fragment screening using biophysical methods. Several fragments binding to this site inhibit ubiquitin conjugation in vitro