Characterization of the transcriptome of fast and slow muscle myotomal fibres in the pacu (Piaractus mesopotamicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

COP 9 SIGNALOSOME SUBUNIT 6 STABILIZES COP1, A NOVEL E3 UBIQUITIN LIGASE FOR 14-3-3σ

14-3-3σ, a gene upregulated by p53 in response to DNA damage, exists as part of a positive-feedback loop which activates p53 and is a human cancer epithelial marker downregulated in various cancer types. 14-3-3σ levels are critical for maintaining p53 activity in response to DNA damage and regulating signal mediator such as Akt. Here, we identify Mammalian Constitutive Photomorphogenic 1 (COP1) as a novel E3 ubiquitin ligase for targeting 14-3-3σ through proteasome degradation. We show for the first time that COP9 signalosome subunit 6 (CSN6) associates with COP1 and is involved in 14-3-3σ ubiquitin-mediated degradation. Mechanistic studies show that CSN6 expression leads to stabilization of COP1 through reducing COP1 self-ubiquitination and decelerating COP1’s turnover rate. We also show that CSN6-mediated 14-3-3σ ubiquitination is compromised when COP1 is knocked down. Thus, CSN6 mediates 14-3-3σ ubiquitination through enhancing COP1 stability. Subsequently, we show that CSN6 causes 14-3-3σ downregulation, thereby activating Akt and promoting cell survival by suppressing FOXO, an Akt target, transcriptional activity. Also, CSN6 overexpression leads to increased cell growth, transformation and promotes tumorigenicity. Significantly, 14-3-3σ expression can correct the abnormalities mediated by CSN6 expression. These data suggest that the CSN6-COP1 axis is involved in 14-3-3σ degradation, and that deregulation of this axis will promote cell growth and tumorigenicity.

SUMO-SPECIFIC PROTEASE 1 CONTROLS LYMPHOID DEVELOPMENT THROUGH REGULATION OF SUMOYLATION/ACETYLATION SWITCH IN STAT5

SUMOylation has emerged as an important regulatory mechanism for protein function. SUMO-specific proteases (SENPs) are essential for removing SUMO from conjugated proteins in many different systems, but the physiological functions of SENPs are poorly understood. STAT5 (Signal Transducer and Activator of Transcription 5) plays a critical role in the development of lymphoid cells. However, it is not known whether STAT5 is regulated by the SUMOylation pathway. Here, we showed that SUMOylated STAT5 is accumulated in SENP1-/- lymphoid precursors. SENP1 deficiency results in severe defects in early T and B cell development, similar to that observed in mice harboring a complete inactivation of STAT5. Because STAT5 is SUMOylated and acetylated at the same lysine residue, SENP1 deficiency blocks STAT5 in the SUMOylation state, resulting in diminished STAT5 acetylation and phosphorylation, and defective lymphoid development. Thus, our results reveal a novel function of SENP1 in the regulation of early lymphoid development via an acetylation/SUMOylation switch in STAT5.

Staying under the radar - the multifunctional LANA Protein

Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposi´s Sarcoma associated herpesvirus (KSHV), first discovered in Kaposi´s Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-κB and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses.

In vivo characterization of the circadian clock output gene usp2

Life on earth is subject to the repeated change between day and night periods. All organisms that undergo these alterations have to anticipate consequently the adaptation of their physiology and possess an endogenous periodicity of about 24 hours called circadian rhythm from the Latin circa (about) and diem (day). At the molecular level, virtually all cells of an organism possess a molecular clock which drives rhythmic gene expression and output functions. Besides altered rhythmicity in constant conditions, impaired clock function causes pathophysiological conditions such as diabetes or hypertension. These data unveil a part of the mechanisms underlying the well-described epidemiology of shift work and highlight the function of clock-driven regulatory mechanisms. The post-translational modification of proteins by the ubiquitin polypeptide is a central mechanism to regulate their stability and activity and is capital for clock function. Similarly to the majority of biological processes, it is reversible. Deubiquitylation is carried out by a wide variety of about ninety deubiquitylating enzymes and their function remains poorly understood, especially in vivo. This class of proteolytic enzymes is parted into five families including the Ubiquitin-Specific Proteases (USP), which is the most important with about sixty members. Among them, the Ubiquitin-Specific Protease 2 (Usp2) gene encodes two protein isoforms, USP2-45 and USP2-69. The first is ubiquitously expressed under the control of the circadian clock and displays all features of core clock genes or its closest outputs effectors. Additionally, Usp2-45 was also found to be induced by the mineralocorticoid hormone aldosterone and thought to participate in Na+ reabsorption and blood pressure regulation by Epithelial Na+ Channel ENaC in the kidneys. During my thesis, I aimed to characterize the role of Usp2 in vivo with respect to these two areas, by taking advantage of a total constitutive knockout mouse model. In the first project I aimed to validate the role of USP2-45 in Na+ homeostasis and blood pressure regulation by the kidneys. I found no significant alterations of diurnal Na+ homeostasis and blood pressure in these mice, indicating that Usp2 does not play a substantial role in this process. In urine analyses, we found that our Usp2-KO mice are actually hypercalciuric. In a second project, I aimed to understand the causes of this phenotype. I found that the observed hypercalciuria results essentially from intestinal hyperabsorption. These data reveal a new role for Usp2 as an output effector of the circadian clock in dietary Ca2+ metabolism in the intestine.

USP2-45 represses aldosterone mediated responses by decreasing mineralocorticoid receptor availability.

BACKGROUND/AIMS: Ligand activation of the mineralocorticoid receptor (MR) induces several post-translational modifications (PTMs). Among the different PTMs, MR is known to be dynamically ubiquitylated with impact on its stability and transcriptional activity. Previously, we have shown that MR is monoubiquitylated at the basal state and that aldosterone stimulation induces monoubiquitylation removal prompting polyubiquitin-dependent destabilization of the receptor and proteasomal degradation. This study investigated the role of the aldosterone induced ubiquitin-specific protease USP2-45 on the ubiquitylation state of MR. METHODS: Renal epithelial cells M1 were co-transfected with MR with or without wild-type or inactive USP2-45. The association of MR with USP2-45 or TSG101 as well as MR ubiquitylation state were determined by immunoprecipitation and immunoblotting. MR transcriptional activity was assessed via a luciferase reporter gene. RESULTS: We show that USP2-45 is able to bind MR and, similarly to aldosterone, induce MR monoubiquitylation removal, disruption of MR/TSG101 association and destabilization of MR at protein level. CONCLUSION: This study provides a novel role for USP2-45 by playing a pivotal role in the regulation of the ubiquitylation state of MR and reveals the existence of a negative feedback loop for limiting the aldosterone induced response.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels

KCNQ1 (Kv7.1), together with its KCNE β subunits, plays a pivotal role both in the repolarization of cardiac tissue and in water and salt transport across epithelial membranes. Nedd4/Nedd4-like (neuronal precursor cell-expressed developmentally downregulated 4) ubiquitin-protein ligases interact with the KCNQ1 potassium channel through a PY motif located in the C terminus of KCNQ1. This interaction induces ubiquitylation of KCNQ1, resulting in a reduced surface density of the channel. It was reported recently that the epithelial sodium channel is regulated by the reverse process-deubiquitylation-mediated by USP2 (ubiquitin-specific protease 2).

Brooke-Spiegler Syndrome - an underrecognized cause of multiple familial scalp tumors: report of a new germline mutation

BACKGROUND: Brooke-Spiegler syndrome (BSS) is probably an underdiagnosed genodermatosis that predisposes for the development of cylindromas, spiradenomas and trichoepitheliomas mainly of the head and neck. Wide phenotypic variability regarding the number and type of lesions can be observed within a family. Mutations of the CYLD gene are identified in the vast majority of cases and play a key role in BSS pathogenesis. MAIN OBSERVATIONS: Two first degree relatives with numerous erythematous telangiectatic nodules of the scalp present for decades, with recurring tendency regardless the multiple previous excisions. Histopathological review of the lesions revealed predominantly "spiradenocylindromas" in the proband and cylindromas in her sister. The suspicion of BSS was confirmed after detection of a new nonsense germline mutation of CYLD (c.1783C>T pGln 595*) in the proband. CONCLUSIONS: BSS diagnosis can be challenging and is based on clinical-pathological correlation, positive familial association and identification of CYLD mutations. CYLD exerts antineoplastic effects by downregulating intracellular NF-κB signalling pathways. The reported mutation affecting the ubiquitin-specific protease domain leads to a truncated and catalytically inactive enzyme. Despite the expanding list of CYLD mutations no firm genotype-phenotype correlation is known so far. Early recognition and treatment of BSS avoid disfiguring changes like "turban tumor".

Brooke-Spiegler Syndrome - an underrecognized cause of multiple familial scalp tumors: report of a new germline mutation

BACKGROUND: Brooke-Spiegler syndrome (BSS) is probably an underdiagnosed genodermatosis that predisposes for the development of cylindromas, spiradenomas and trichoepitheliomas mainly of the head and neck. Wide phenotypic variability regarding the number and type of lesions can be observed within a family. Mutations of the CYLD gene are identified in the vast majority of cases and play a key role in BSS pathogenesis. MAIN OBSERVATIONS: Two first degree relatives with numerous erythematous telangiectatic nodules of the scalp present for decades, with recurring tendency regardless the multiple previous excisions. Histopathological review of the lesions revealed predominantly "spiradenocylindromas" in the proband and cylindromas in her sister. The suspicion of BSS was confirmed after detection of a new nonsense germline mutation of CYLD (c.1783C>T pGln 595*) in the proband. CONCLUSIONS: BSS diagnosis can be challenging and is based on clinical-pathological correlation, positive familial association and identification of CYLD mutations. CYLD exerts antineoplastic effects by downregulating intracellular NF-κB signalling pathways. The reported mutation affecting the ubiquitin-specific protease domain leads to a truncated and catalytically inactive enzyme. Despite the expanding list of CYLD mutations no firm genotype-phenotype correlation is known so far. Early recognition and treatment of BSS avoid disfiguring changes like "turban tumor".

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

Linking the SUMO protease SENP5 to neutrophil differentiation of AML cells

In an mRNA profiling screen performed to unveil novel mechanisms of leukemogenesis, we found that the sentrin-specific protease 5 (SENP5) was significantly repressed in clinical acute myeloid leukemia when compared to healthy neutrophil samples. SENP5 is an enzyme that targets and cleaves small ubiquitin-like modifier (SUMO) residues from SUMOylated proteins. Further investigation with AML neutrophil differentiation cell models showed increased SENP5 expression upon induction of differentiation; in contrast, knocking down SENP5 resulted in significantly attenuated neutrophil differentiation. Our results support a new role of SENP5 in AML pathology, and in particular in the neutrophil differentiation of myeloid leukemic cells.

Using ubiquitin to follow the metabolic fate of a protein.

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.