Mice carrying ubiquitin-specific protease 2 (Usp2) gene inactivation maintain normal sodium balance and blood pressure

Ubiquitylation plays an important role in the control of Na⁺ homeostasis by the kidney. It is well established that the epithelial Na⁺ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney and is also a circadian output gene. In heterologous expression systems, USP2-45 binds to ENaC, deubiquitylates it, and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na⁺ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO mice did not show any differences from wild-type littermates with respect to the diurnal control of Na⁺ or K⁺ urinary excretion and plasma levels either on a standard diet or after acute and chronic changes to low- and high-Na⁺ diets, respectively. Moreover, they had similar aldosterone levels on either a low- or high-Na⁺ diet. Blood pressure measurements using telemetry did not reveal variations compared with control mice. Usp2-KO mice did not display alterations in expression of genes involved in sodium homeostasis or the ubiquitin system, as evidenced by transcriptome analysis in the kidney. Our data suggest that USP2 does not play a primary role in the control of Na⁺ balance or blood pressure.

Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

In pneumococcal meningitis a novel water-soluble inhibitor of matrix metalloproteinases and TNF-alpha converting enzyme attenuates seizures and injury of the cerebral cortex

Matrix metalloproteinases (MMPs) and TNF-alpha converting enzyme (TACE) contribute to the pathophysiology of bacterial meningitis. To date, MMP-inhibitors studied in models of meningitis were compromised by their hydrophobic nature. We investigated the pharmacokinetics and the effect of TNF484, a water-soluble hydroxamate-based inhibitor of MMP and TACE, on disease parameters and brain damage in a neonatal rat model of pneumococcal meningitis. At 1 mg/kg q6h TNF484 reduced soluble TNF-alpha and the collagen degradation product hydroxyproline in the cerebrospinal fluid. Clinically, TNF484 attenuated the incidence of seizures and was neuroprotective in the cortex. Water-soluble MMP-inhibitors may hold promise in the therapy of bacterial meningitis.

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease

OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (pFDR=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.

Swiss national guideline for reimbursement of enzyme replacement therapy in late-onset Pompe disease.

Glycogen storage disease type II is a rare multi-systemic disorder characterised by an intracellular accumulation of glycogen due a mutation in the acid alpha glucosidase (GAA) gene. The level of residual enzyme activity, the genotype and other yet unknown factors account for the broad variation of the clinical phenotype. The classical infantile form is characterised by severe muscle hypotonia and cardiomyopathy leading to early death. The late-onset form presents as a limb girdle myopathy with or without pulmonary dysfunction. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) in infants is life saving. In contrast, therapeutic efficacy of rhGAA in the late-onset form is modest. High expenses of rhGAA, on-going infusions and poor pharmacokinetic efficacy raised a discussion of the cost effectiveness of ERT in late-onset Pompe disease in Switzerland. This discussion was triggered by a Swiss federal court ruling which confirmed the reluctance of a health care insurer not to reimburse treatment costs in a 67-year-old female suffering from Pompe disease. As a consequence of this judgement ERT was stopped by all insurance companies in late-onset Pompe patients in Switzerland regardless of their clinical condition. Subsequent negotiations lead to the release of a national guideline of the management of late-onset Pompe disease. Initiation and limitation of ERT is outlined in a national Pompe registry. Reimbursement criteria are defined and individual efficacy of ERT with rhGAA is continuously monitored.

A single localized dose of enzyme-responsive hydrogel improves long-term survival of a vascularized composite allograft

Currently, systemic immunosuppression is used in vascularized composite allotransplantation (VCA). This treatment has considerable side effects and reduces the quality of life of VCA recipients. We loaded the immunosuppressive drug tacrolimus into a self-assembled hydrogel, which releases the drug in response to proteolytic enzymes that are overexpressed during inflammation. A one-time local injection of the tacrolimus-laden hydrogel significantly prolonged graft survival in a Brown Norway-to-Lewis rat hindlimb transplantation model, leading to a median graft survival of >100 days compared to 33.5 days in tacrolimus only-treated recipients. Control groups with no treatment or hydrogel only showed a graft survival of 11 days. Histopathological evaluation, including anti-graft antibodies and complement C3, revealed significantly reduced immune responses in the tacrolimus-hydrogel group compared with tacrolimus only. In conclusion, a single-dose local injection of an enzyme-responsive tacrolimus-hydrogel is capable of preventing VCA rejection for >100 days in a rat model and may offer a new approach for immunosuppression in VCA.

Current role of enzyme analysis for urea cycle disorders

Urea cycle disorders (UCD) are due to defects of any of its six enzymes or two transporters. The definitive diagnosis of defects of the three mitochondrial enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase I (CPS1) and ornithine transcarbamylase (OTC) depends on either molecular mutation analysis or measurement of enzyme activity, whereas the diagnosis of deficiencies of the three cytosolic enzymes argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase I (ARG1) is usually straightforward, based on marker metabolites. Enzyme assays for all UCD have been used since their first description, for disease confirmation and in some instances even for prenatal diagnosis. The genetic bases of the UCD have only been unraveled from the 1980s; the last gene cloned being the NAGS gene in 2002. In this review we discuss the enzymatic assays for all urea cycle enzymes from a historical perspective, their potential and drawbacks, and the current role of enzymatic analysis in UCD in general.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

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.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

CYP17A1 Enzyme Activity Is Linked to Ambulatory Blood Pressure in a Family-Based Population Study

BACKGROUND Genome-wide association studies have linked CYP17A1 coding for the steroid hormone synthesizing enzyme 17α-hydroxylase (CYP17A1) to blood pressure (BP). We hypothesized that the genetic signal may translate into a correlation of ambulatory BP (ABP) with apparent CYP17A1 activity in a family-based population study and estimated the heritability of CYP17A1 activity. METHODS In the Swiss Kidney Project on Genes in Hypertension, day and night urinary excretions of steroid hormone metabolites were measured in 518 participants (220 men, 298 women), randomly selected from the general population. CYP17A1 activity was assessed by 2 ratios of urinary steroid metabolites: one estimating the combined 17α-hydroxylase/17,20-lyase activity (ratio 1) and the other predominantly 17α-hydroxylase activity (ratio 2). A mixed linear model was used to investigate the association of ABP with log-transformed CYP17A1 activities exploring effect modification by urinary sodium excretion. RESULTS Daytime ABP was positively associated with ratio 1 under conditions of high, but not low urinary sodium excretion (P interaction <0.05). Ratio 2 was not associated with ABP. Heritability estimates (SE) for day and night CYP17A1 activities were 0.39 (0.10) and 0.40 (0.09) for ratio 1, and 0.71 (0.09) and 0.55 (0.09) for ratio 2 (P values <0.001). CYP17A1 activities, assessed with ratio 1, were lower in older participants. CONCLUSIONS Low apparent CYP17A1 activity (assessed with ratio 1) is associated with elevated daytime ABP when salt intake is high. CYP17A1 activity is heritable and diminished in the elderly. These observations highlight the modifying effect of salt intake on the association of CYP17A1 with BP.

A novel CYP17A1 deletion causes a functional knockout of the steroid enzyme 17-hydroxylase and 17,20-lyase in a Turkish family and illustrates the precise role of the CYP17A1 gene

A novel homozygous long-range deletion of the CYP17A1 gene abolished protein expression and caused the severest form of 17-hydroxylase deficiency in one kindred of a Turkish family. The affected subjects presented with 46,XY sex reversal and 46,XX lack of pubertal development as well as severe hypertension.

Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.

BACKGROUND Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers. OBJECTIVE The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces. METHODS Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated. RESULTS Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations. CONCLUSIONS The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.