Ubiquitin-Specific Protease 25 Functions in Endoplasmic Reticulum-Associated Degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome

Myosin VIIA, Important for Human Auditory Function, Is Necessary for Drosophila Auditory Organ Development

BACKGROUND: Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research

Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans

BACKGROUND: The Usher syndrome (USH) is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively. PRINCIPAL FINDINGS: Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche. CONCLUSIONS: Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease

Splice Isoforms of the Polyglutamine Disease Protein Ataxin-3 Exhibit Similar Enzymatic yet Different Aggregation Properties

Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively). In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5′ variants and both of the known 3′ ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity

Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology

Neural Representations of Airflow in Drosophila Mushroom Body

The Drosophila mushroom body (MB) is a higher olfactory center where olfactory and other sensory information are thought to be associated. However, how MB neurons of Drosophila respond to sensory stimuli other than odor is not known. Here, we characterized the responses of MB neurons to a change in airflow, a stimulus associated with odor perception. In vivo calcium imaging from MB neurons revealed surprisingly strong and dynamic responses to an airflow stimulus. This response was dependent on the movement of the 3rd antennal segment, suggesting that Johnston's organ may be detecting the airflow. The calyx, the input region of the MB, responded homogeneously to airflow on. However, in the output lobes of the MB, different types of MB neurons responded with different patterns of activity to airflow on and off. Furthermore, detailed spatial analysis of the responses revealed that even within a lobe that is composed of a single type of MB neuron, there are subdivisions that respond differently to airflow on and off. These subdivisions within a single lobe were organized in a stereotypic manner across flies. For the first time, we show that changes in airflow affect MB neurons significantly and these effects are spatially organized into divisions smaller than previously defined MB neuron types

Ataxin-3 Plays a Role in Mouse Myogenic Differentiation through Regulation of Integrin Subunit Levels

BACKGROUND: During myogenesis several transcription factors and regulators of protein synthesis and assembly are rapidly degraded by the ubiquitin-proteasome system (UPS). Given the potential role of the deubiquitinating enzyme (DUB) ataxin-3 in the UPS, and the high expression of the murine ataxin-3 homolog in muscle during embryogenesis, we sought to define its role in muscle differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence analysis, we found murine ataxin-3 (mATX3) to be highly expressed in the differentiated myotome of E9.5 mouse embryos. C2C12 myoblasts depleted of mATX3 by RNA interference exhibited a round morphology, cell misalignment, and a delay in differentiation following myogenesis induction. Interestingly, these cells showed a down-regulation of alpha5 and alpha7 integrin subunit levels both by immunoblotting and immunofluorescence. Mouse ATX3 was found to interact with alpha5 integrin subunit and to stabilize this protein by repressing its degradation through the UPS. Proteomic analysis of mATX3-depleted C2C12 cells revealed alteration of the levels of several proteins related to integrin signaling. CONCLUSIONS: Ataxin-3 is important for myogenesis through regulation of integrin subunit levels.This work was financed by the Fundacao para a Ciencia e a Tecnologia (FCT) (POCI/SAU-MMO/60412/2002) and by National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) grant RO1 NS038712 to HLP. MCC, FB, AJR, and RJT were supported by the FCT fellowships (SFRH/BD/9759/2003 and SFRH/BPD/28560/2006), (SFRH/BPD/17368/2004), (SFRH/BD/17066/2004), (SFRH/BD/29947/2006), respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

PTMs in Conversation: Activity and Function of Deubiquitinating Enzymes Regulated via Post-Translational Modifications

Deubiquitinating enzymes (DUBs) constitute a diverse protein family and their impact on numerous biological and pathological processes has now been widely appreciated. Many DUB functions have to be tightly controlled within the cell, and this can be achieved in several ways, such as substrate-induced conformational changes, binding to adaptor proteins, proteolytic cleavage, and post-translational modifications (PTMs). This review is focused on the role of PTMs including monoubiquitination, sumoylation, acetylation, and phosphorylation as characterized and putative regulative factors of DUB function. Although this aspect of DUB functionality has not been yet thoroughly studied, PTMs represent a versatile and reversible method of controlling the role of DUBs in biological processes. In several cases PTMs might constitute a feedback mechanism insuring proper functioning of the ubiquitin proteasome system and other DUB-related pathways

The ubiquitin proteasome system in neuropathology

The ubiquitin proteasome system (UPS) orchestrates the turnover of innumerable cellular proteins. In the process of ubiquitination the small protein ubiquitin is attached to a target protein by a peptide bond. The ubiquitinated target protein is subsequently shuttled to a protease complex known as the 26S proteasome and subjected to degradative proteolysis. The UPS facilitates the turnover of proteins in several settings. It targets oxidized, mutant or misfolded proteins for general proteolytic destruction, and allows for the tightly controlled and specific destruction of proteins involved in development and differentiation, cell cycle progression, circadian rhythms, apoptosis, and other biological processes. In neuropathology, alteration of the UPS, or mutations in UPS target proteins may result in signaling abnormalities leading to the initiation or progression of tumors such as astrocytomas, hemangioblastomas, craniopharyngiomas, pituitary adenomas, and medulloblastomas. Dysregulation of the UPS may also contribute to tumor progression by perturbation of DNA replication and mitotic control mechanisms, leading to genomic instability. In neurodegenerative diseases caused by the expression of mutant proteins, the cellular accumulation of these proteins may overload the UPS, indirectly contributing to the disease process, e.g., sporadic Parkinsonism and prion diseases. In other cases, mutation of UPS components may directly cause pathological accumulation of proteins, e.g., autosomal recessive Parkinsonism and spinocerebellar ataxias. Defects or dysfunction of the UPS may also underlie cognitive disorders such as Angelman syndrome, Rett syndrome and autism, and muscle and nerve diseases, e.g., inclusion body myopathy and giant axon neuropathy. This paper describes the basic biochemical mechanisms comprising the UPS and reviews both its theoretical and proven involvement in neuropathological diseases. The potential for the UPS as a target of pharmacological therapy is also discussed