Mutations in Parkin, an E3 ubiquitin ligase that regulates protein turnover, represent one of the major causes of familial Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons and impaired mitochondrial functions. The underlying mechanism by which pathogenic Parkin mutations induce mitochondrial abnormality is not fully understood. Here, we demonstrate that Parkin interacts with and subsequently ubiquitinates dynamin-related protein 1 (Drp1), for promoting its proteasome-dependent degradation. Pathogenic mutation or knockdown of Parkin inhibits the ubiquitination and degradation of Drp1, leading to an increased level of Drp1 for mitochondrial fragmentation. These results identify Drp1 as a novel substrate of Parkin and suggest a potential mechanism linking abnormal Parkin expression to mitochondrial dysfunction in the pathogenesis of Parkinson disease. Parkinson disease (PD)4 is one of the most common neurodegenerative diseases affecting over 2% populations over 65 years of age. It is classically characterized by the loss of dopaminergic neurons that project from the midbrain substantia nigra to the striatum (1, 2). Although the loss of dopaminergic neurons is responsible for the symptom of movement disorder in PD, it is now clear that other types of neurons throughout the brain are also affected in the disease (3, 4). The identification of genes linking to PD has greatly advanced our understanding of the molecular pathogenesis of the disease (5-8). Mutations in Parkin represent one of major causes for early onset of familial PD (9 -11). Parkin is an E3 ubiquitin ligase that contains two ring finger domains (12-15). A handful of substrates have been identified, including Parkin itself and CDCrel-1, synphilin-1, Pael-R, glycosylated ␣-synuclein, FBP1 (far upstream elementbinding protein 1), and the RNA-processing protein subunit p38/AIMP2 (16 -19). A putative mechanism by which mutations of Parkin cause PD would be abnormal accumulation and aggregation of the above substrates due to insufficient E3 ligase activity for ubiquitin-proteasome-dependent protein turnover (18,20,21). Surprisingly, only p38/AIMP2 and FBP1 were found to be accumulated in the brain samples of PD patients or in Parkin knock-out mice (16,17,19). Even though a number of the putative substrates have been identified, the causative link between these substrates and the PD pathogenesis remains not fully understood.Over the past few decades, accumulating evidence has suggested that mitochondrial dysfunction and the resulting oxidative damage are associated with PD. This is supported by a large number of reports demonstrating impaired mitochondrial functions in PD patients (22)(23)(24)(25)(26). Mitochondria undergo frequent fission, fusion, and redistribution throughout the cytoplasm in response to the energy needs (27,28). Either disruption of the fusion process or enhancement of the fission process renders the normal, tubular network of mitochondria to fragment into short rods or spheres (29). Abnormal...

Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

The familial cylindromatosis tumor suppressor CYLD is known to contain three cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, which exist in a number of microtubule-binding proteins and are responsible for their association with microtubules. However, it remains elusive whether CYLD interacts with microtubules and, if so, whether the interaction is mediated by the CAP-Gly domains. In this study, our data demonstrate that CYLD associates with microtubules both in cells and in vitro, and the first CAP-Gly domain of CYLD is mainly responsible for the interaction. Knockdown of cellular CYLD expression dramatically delays microtubule regrowth after nocodazole washout, indicating an activity for CYLD in promoting microtubule assembly. Our data further demonstrate that CYLD enhances tubulin polymerization into microtubules by lowering the critical concentration for microtubule assembly. In addition, we have identified by wound healing assay a critical role for CYLD in mediating cell migration and found that its first CAP-Gly domain is required for this activity. Thus CYLD joins a growing list of CAP-Gly domain-containing proteins that regulate microtubule dynamics and function.

Promoting Complement (C) activation may enhance immunological mechanisms of anti-tumor antibodies for tumor destruction. However, C activation components, such as C5a, trigger inflammation which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector (CV), or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared to CV tumors. NK cells and macrophages infiltrated C5a expressing tumors with significantly greater frequency while VEGF, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1+CD11b+ myeloid cells in spleen and overall decreased CD4+ and CD8+ T cells in tumor, tumor-draining lymph nodes (TDLN), and spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ-producing CD4+ and CD8+ T cells in spleen and TDLN. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.

Cilia are hair-like organelles extending from the cell surface with important sensory and motility functions. Ciliary defects can result in a wide range of human diseases known as ciliopathies. However, the molecular mechanisms controlling ciliogenesis remain poorly defined. Here we show that cylindromatosis (CYLD), a tumor suppressor protein harboring deubiquitinase activity, plays a critical role in the assembly of both primary and motile cilia in multiple organs. CYLD knockout mice exhibit polydactyly and various ciliary defects, such as failure in basal body anchorage and disorganization of basal bodies and axenomes. The ciliary function of CYLD is partially attributed to its deconjugation of the polyubiquitin chain from centrosomal protein of 70 kDa (Cep70), a requirement for Cep70 to interact with γ-tubulin and localize at the centrosome. In addition, CYLD-mediated inhibition of histone deacetylase 6 (HDAC6), which promotes tubulin acetylation, constitutes another mechanism for the ciliary function of CYLD. Small-molecule inhibitors of HDAC6 could partially rescue the ciliary defects in CYLD knockout mice. These findings highlight the importance of protein ubiquitination in the modulation of ciliogenesis, identify CYLD as a crucial regulator of this process, and suggest the involvement of CYLD deficiency in ciliopathies.