Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

DUB-1, a deubiquitinating enzyme with growth-suppressing activity.

Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s).

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.

Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor (PIF)

Loss of skeletal muscle is a major factor in the poor survival of patients with cancer cachexia. This study examines the mechanism of catabolism of skeletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intravenous administration of PIF to normal mice produced a rapid decrease in body weight (1.55 ± 0.12 g in 24 h) that was accompanied by increased mRNA levels for ubiquitin, the Mr 14 000 ubiquitin carrier-protein, E2, and the C9 proteasome subunit in gastrocnemius muscle. There was also increased protein levels of the 20S proteasome core and 19S regulatory subunit, detectable by immunoblotting, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. An increased protein catabolism was also seen in C2C12 myoblasts within 24 h of PIF addition with a bell-shaped dose-response curve and a maximal effect at 2-4 nM. The enhanced protein degradation was attenuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lactacystin. Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis showed a dose-related increase in expression of MSSI, an ATPase that is a regulatory subunit of the proteasome, with a dose-response curve similar to that for protein degradation. These results confirm that PIF acts directly to stimulate the proteasome pathway in muscle cells and may play a pivotal role in protein catabolism in cancer cachexia. © 2001 Cancer Research Campaign.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Bioactive beads for local sensing of proteases in 3D engineered tissues

Dissertação para obtenção do Grau de Doutor em Bioengenharia (MIT)

Targeting B-cell lymphomas with inhibitors of the MALT1 paracaspase.

The paracaspase MALT1 is an Arg-specific protease that cleaves multiple substrates to promote lymphocyte proliferation and survival. The catalytic activity of MALT1 is normally tightly regulated by antigen receptor triggering, which promotes MALT1 activation by its inducible monoubiquitination-dependent dimerization. Constitutive MALT1 activity is a hallmark of specific subsets of B-cell lymphomas, which are characterized by chromosomal translocations or point mutations that activate MALT1 or its upstream regulators. Recent findings suggest that such lymphomas may be sensitive to treatment with MALT1 inhibitors. Here we review recent progress in the understanding of MALT1 function and regulation, and the development of small molecule MALT1 inhibitors for therapeutic applications.

Detection and measurement of paracaspase MALT1 activity.

The paracaspase MALT1 is a Cys-dependent, Arg-specific protease that plays an essential role in the activation and proliferation of lymphocytes during the immune response. Oncogenic activation of MALT1 is associated with the development of specific forms of B-cell lymphomas. Through specific cleavage of its substrates, MALT1 controls various aspects of lymphocyte activation, including the activation of transcriptional pathways, the stabilization of mRNAs, and an increase in cellular adhesion. In lymphocytes, the activity of MALT1 is tightly controlled by its inducible monoubiquitination, which promotes the dimerization of MALT1. Here, we describe both in vitro and in vivo assays that have been developed to assess MALT1 activity.

Marine04 marine radiocarbon age calibration, 0-26 cal kyr BP

New radiocarbon calibration curves, IntCal04 and Marine04, have been constructed and internationally ratified to replace the terrestrial and marine components of IntCal98. The new calibration data sets extend an additional 2000 yr, from 0-26 cal kyr BP (Before Present, 0 cal. BP = AD 1950), and provide much higher resolution, greater precision, and more detailed structure than IntCal98. For the Marine04 curve, dendrochronologically-dated tree-ring samples, converted with a box diffusion model to marine mixed-layer ages, cover the period from 0-10.5 call kyr BR Beyond 10.5 cal kyr BP, high-resolution marine data become available from foraminifera in varved sediments and U/Th-dated corals. The marine records are corrected with site-specific C-14 reservoir age information to provide a single global marine mixed-layer calibration from 10.5-26.0 cal kyr BR A substantial enhancement relative to IntCal98 is the introduction of a random walk model, which takes into account the uncertainty in both the calendar age and the C-14 age to calculate the underlying calibration curve (Buck and Blackwell, this issue). The marine data sets and calibration curve for marine samples from the surface mixed layer (Marine04) are discussed here. The tree-ring data sets, sources of uncertainty, and regional offsets are presented in detail in a companion paper by Reimer et al. (this issue).

Relação entre padrão comportamental, estágios do ciclo de muda e atividade de enzimas digestivas proteolíticas em juvenis do camarão marinho Litopenaeus vannamei (Crustacea: Penaeidae)

Shrimp farming in Brazil is a consolidated activity, having brought economical and social gains to several states with the largest production concentrated in the northeast. This fact is also reflected in higher feed intake, necessitating a more efficient feed management. Currently, management techniques already foresee food loss due to molting. In this sense, studies relating shrimp s digestive physiology, molting physiology and behavioral response of shrimp feed can optimize the feed management. Thus, our study aimed to evaluate the behavioral response of the marine shrimp L. vannamei (Crustacea: Penaeidae) in accordance with the stages of moulting cycle and feeding schedules based on higher or lower activity of proteolytic digestive enzymes; also, to investigate the influence of feeding schedule on hepatosomatic index and non-specific and specific protease activity (trypsin). Experiments were carried out at the Laboratory of Shrimp Behavioral Studies at UFRN in partnership with the Laboratory of Enzimology UFPE. Juveniles of L. vannamei weighting 5.25 g (+ 0.25 g) were kept in aquaria at a density of 33 shrimp m -2. In the first experiment, shrimp were fed in the light phase or in the dark phase for 8 days; in the ninth day, the animals were observed for 15 minutes every hour during the 12 hours of each phase of the photoperiod. We recorded the frequency of inactivity, exploration, food intake, burrowing, swimming and crawling behavior. At the end of the 12th observation session, the shrimp were sacrified and classified by the method of setogenesis in the molt cycle stages A, B, C, D0, D1, D2 or D3. We found that the shrimp in A stage show high levels of inactivity. Moreover, the frequency of food intake was very low. The shrimp in D3 stage also had low food intake and high inactivity associated with elevated frequencies of burrowing. In the second experiment, shrimp were kept in physiological acclimation to experimental conditions for 28 days, distributed in 12 treatments in the light phase and 12 treatments in the dark phase. In the end, the animals were sacrified and dissected to assess non-specific and specific protease activity (trypsin) activity. In general, these parameters did not vary among animals fed in the light phase and those fed in the dark phase. However, significant differences were found in the activity of specific and nonspecific proteases in relation to food treatment. In the light phase, the major proteolytic activities converged to 10 hours after the start of the light phase, while the lowest activities converged to 6 hours after the beginning of this phase. In the dark phase, the highest enzyme activity converged to 12 hours after the onset of phase, while the lowest activities converged to 3 hours after the onset of phase. In the third experiment, we sought to evaluate the behavioral responses of shrimp in relation to dietary treatments based on higher or lower activity of proteolytic enzymes, considering the results of the second experiment. The behavioral categories observed were the same as the ones in the first experiment, with observations of 30 minutes (15min before and 15min after food supply). We found variation in behavioral responses as a function of the treatments, with greater intake of food in shrimp fed during the period of greatest activity of proteolytic enzymes, in the light phase. Thus we see that periodic events associated with the shrimp s physiology interfere in their behavioral responses, revealing situations that are more adjustable to the provision of food, and consequently optimizing feeding management

Uso de complexo enzimático em dietas para tilápia do Nilo: digestibilidade, atividade enzimática, desempenho produtivo e parâmetros fisiológicos

Pós-graduação em Zootecnia - FCAV

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^