Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.</p

Quadrant Analysis of Shear Events in Open Channel Flows Over Mobile and Immobile Hydraulically Rough Beds

Sediment overfeeding may induce important changes in the structure of the near-bed region of gravel-bed river flows, mainly in what concerns exchange of momentum and mass between the flow within the riverbed roughness elements and the flow in the upper regions. Although the turbulent structure of flows over gravel- bed rivers is object of several previous studies, it is not well-known how statistics characterizing coherent turbulence events responsible by the generation of turbulent shear stresses are affected by bed load transport in flows over hydraulically rough beds with low relative submergence. This study is aimed at bridging this research gap. It is based on two-dimensional instantaneous velocity data, in the stream-wise and vertical directions, acquired with Particle Image Velocimetry in a laboratory flume. Two tests simulated framework gravel beds with sand matrixes, one of which fed with sand at near capacity conditions. The framework, immobile under the imposed flow conditions, consists of coarse gravel whose diameters range between 0.5 cm and 7 cm. Matrix and imposed sand feature a median diameter of 0.9 mm. For both tests, the quadrant threshold analysis technique was employed and transported momentum were analyzed and discussed in what concerns their intensity distribution for events in the four quadrants, and for several positions within the flow. It is shown that under mobile bed conditions, sweeps are dominant in the turbulence production in the pythmenic region of the flow. In the outer region of the flow, this is independent from the channel bed; in the overlapping intermediate layer, between the inner region and the pythmenic region, the flow characteristics depend on the position in relation to the crests and troughs of the bed

Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity

Parkinson’s disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators

Discovery and characterization of noncanonical E2-conjugating enzymes

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.</p