Capturing the catalytic intermediates of parkin ubiquitination.

Parkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate.

Using Förster Resonance Energy Transfer (FRET) to Understand the Ubiquitination Landscape.

Förster resonance energy transfer (FRET) is a fluorescence technique that allows quantitative measurement of protein interactions, kinetics and dynamics. This review covers the use of FRET to study the structures and mechanisms of ubiquitination and related proteins. We survey FRET assays that have been developed where donor and acceptor fluorophores are placed on E1, E2 or E3 enzymes and ubiquitin (Ub) to monitor steady-state and real-time transfer of Ub through the ubiquitination cascade.

Role of calcium-sensor proteins in cell membrane repair.

Cell membrane repair is a critical process used to maintain cell integrity and survival from potentially lethal chemical, and mechanical membrane injury. Rapid increases in local calcium levels due to a membrane rupture have been widely accepted as a trigger for multiple membrane-resealing models that utilize exocytosis, endocytosis, patching, and shedding mechanisms. Calcium-sensor proteins, such as synaptotagmins (Syt), dysferlin, S100 proteins, and annexins, have all been identified to regulate, or participate in, multiple modes of membrane repair.

Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn//Sod2 animals did not develop dopamine cell loss in the S.

Distinct phosphorylation signals drive acceptor versus free ubiquitin chain targeting by parkin.

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of the Ubl domain in parkin, both mediated by the OMM kinase, PINK1. How an OMM protein is selected for ubiquitination is unclear.

Mechanism of Zn and Ca Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn, but the molecular mechanisms of this binding are not yet known.

Interactions between the Cell Membrane Repair Protein S100A10 and Phospholipid Monolayers and Bilayers.

Protein S100A10 participates in different cellular mechanisms and has different functions, especially at the membrane. Among those, it forms a ternary complex with annexin A2 and the C-terminal of AHNAK and then joins the dysferlin membrane repair complex. Together, they act as a platform enabling membrane repair.

Acetylated Ubiquitin Modulates the Catalytic Activity of the E1 Enzyme Uba1.

Ubiquitin (Ub) signaling requires the covalent passage of Ub among E1, E2, and E3 enzymes. The choice of E2 and E3 enzymes combined with multiple rounds of the cascade leads to the formation of polyubiquitin chains linked through any one of the seven lysines on Ub. The linkage type and length act as a signal to trigger important cellular processes such as protein degradation or the DNA damage response.

A mechanistic review of Parkin activation.

Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phospho-ubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound.

Recruitment of Ubiquitin within an E2 Chain Elongation Complex.

The ubiquitin (Ub) proteolysis pathway uses an E1, E2, and E3 enzyme cascade to label substrate proteins with ubiquitin and target them for degradation. The mechanisms of ubiquitin chain formation remain unclear and include a sequential addition model, in which polyubiquitin chains are built unit by unit on the substrate, or a preassembly model, in which polyubiquitin chains are preformed on the E2 or E3 enzyme and then transferred in one step to the substrate. The E2 conjugating enzyme UBE2K has a 150-residue catalytic core domain and a C-terminal ubiquitin-associated (UBA) domain.

Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns.

Ubiquitination is a post-translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro. Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion.

The mammalian CTLH complex is an E3 ubiquitin ligase that targets its subunit muskelin for degradation.

The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA.

A subset of calcium-binding S100 proteins show preferential heterodimerization.

The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth.

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.