PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-e-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.ubiquitin-binding protein | ubiquitin ligase | ubiquitination

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Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle.

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Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.

Caveolin 1 (Cav-1) is an oligomeric protein that forms flaskshaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination-and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1.Caveolae are flask-shaped pits in the lipid-rich domains of the plasma membrane. They are structured by an electrondense, membrane-spanning oligomeric protein called caveolin (Cav) 2 and have been implicated in various cellular activities, including endocytosis and signal transduction (1). The mammalian Cav family consists of three members: Cav-1, Cav-2, and Cav-3. Caveolae mainly comprise either Cav-1 or Cav-3 homooligomers, which, at lower proportions, form hetero-oligomers with Cav-2 (2). Cav-1 and Cav-2 are expressed ubiquitously, whereas Cav-3 is primarily expressed in striated muscle. Cav proteins associate with the plasma membrane through their central regions, leaving the N-and C-terminal regions oriented toward the cytoplasm (3). Newly synthesized Cav monomers, after their insertion into the endoplasmic reticulum membrane, are transported to the Golgi apparatus, where they are oligomerized into SDS-resistant 8S oligomers together with cholesterol. The 8S oligomers are finally transported to the plasma membrane, where they form 70S oligomers and are stabilized by regulatory proteins, such as cavin, to constitute complete caveolae (2, 4).Cav-1 undergoes endocytosis from the plasma membrane and is transported to the lysosome for degradation in a ubiquitination-dependent manner (5). Ubiquitination is a posttranslational modification of...

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