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.

Human herpesvirus 8 (HHV-8) and the etiopathogenesis of Kaposi's sarcoma

OBJECTIVE: To review the current literature on human herpesvirus 8 with particular attention to the aspects related to the etiopathogenesis of Kaposi's sarcoma. MATERIALS AND METHODS: The authors searched original research and review articles on specific aspects of human herpesvirus 8 infection, including virology, epidemiology, transmission, diagnosis, natural history, therapy, and Kaposi's sarcoma etiopathogenesis. The relevant material was evaluated and reviewed. RESULTS: Human herpesvirus 8 is a recently discovered DNA virus that is present throughout the world but with major geographic variation. In the Western world, the virus, transmitted mainly by means of sexual contact, is strongly associated with Kaposi's sarcoma and body cavity-based lymphoma and more controversially with multiple myeloma and other non-proliferative disorders. There is no specific effective treatment, but HIV protease inhibitors may play an indirect role in the clearance of human herpesvirus 8 DNA from peripheral blood mononuclear cells of HIV-infected patients. Human herpesvirus 8 DNA is present in saliva, but there are as yet no documented cases of nosocomial transmission to health care workers. The prevalence of human herpesvirus 8 among health care workers is probably similar to that in the general population. CONCLUSION: Human herpesvirus 8 appears to be, at least in Western Europe and United States, restricted to a population at risk of developing Kaposi's sarcoma. Human herpesvirus 8 certainly has the means to overcome cellular control and immune responses and thus predispose carriers to malignancy, particularly Kaposi's sarcoma. The wide diffusion of Human herpesvirus 8 in classic Kaposi's sarcoma areas appears to represent an important factor in the high incidence of the disease. However, additional co-factors are likely to play a role in the development of Kaposi's sarcoma.

Photochemical synthesis and functional transformations of bicyclic vinyl aziridines

Aziridines, a class of organic compounds containing a three membered heterocycle with a nitrogen atom, are extremely valuable molecules in organic and medicinal chemistry. They are frequently used as versatile precursors in the synthesis of natural products, and many biologically active molecules possess the aziridine moiety. The reactivity of aziridines has been studied, for example, in ring-opening reactions with thiols. However, not much interest seems to be given to reactions of aziridines in aqueous media, despite the numberless advantages of using water as solvent in organic chemistry. The nucleophilic ring-opening reaction of aziridines in aqueous media was here explored. Following the Kaplan aziridine synthetic methodology, in which pyridinium salts undergo a photochemical transformation to give bicyclic vinyl aziridines, new aziridines were synthetized. Their nucleophilic ring-opening reaction in water under physiological conditions was investigated and a range of sulphur, nitrogen, carbon and oxygen nucleophiles tested. Thiols, anilines and azide proved to be good nucleophiles to react with the aziridines, giving the ring-opening product in moderate to good yields. The best results were obtained with thiols, more specifically with cysteine-derived nucleophiles. Preliminary results show that these bicyclic vinyl aziridines can modify calcitonin, a peptide containing two cysteine amino acids residues, grating them the potential to be used in bioconjugation as ligands to cysteine-containing proteins, or even as enzyme inhibitors of, for example, cysteine proteases. Additionally, exploratory investigations suggest that the separation of both enantiomers of the bicyclic vinyl aziridine can be performed by taking advantage of an enzymatic methodology for the resolution of racemic secondary alcohols. Both enantiomers would be highly valuable as precursors in the synthesis of enantiomerically pure molecules, as no other method is currently reported for their separation.

Enzymatic processing of protein-based fibers

Wool and silk are major protein fiber materials used by the textile industry. Fiber protein structure-function relationships are briefly described here, and the major enzymatic processing routes for textiles and other novel applications are deeply reviewed. Fiber biomodification is described here with various classes of enzymes such as protease, transglutaminase, tyrosinase, and laccase. It is expected that the reader will get a perspective on the research done as a basis for new applications in other areas such as cosmetics and pharma.

Digestibilidade aparente dos nutrientes e energia de ração suplementada com enzimas digestivas exógenas para juvenis de tambaqui (Colosssoma macropomum Cuvier, 1818)

O experimento foi conduzido com o objetivo de avaliar o efeito da adição de um compelxo multienzimático exógeno composto de amilase, protease, lipase e celulase, em rações de juvenis de tambaqui, sobre os coeficientes de digestibilidade aparente (CDa) da proteína bruta (PB), extrato etéreo (EE), carboidratos (ENN) e energia bruta (EB). O delineamento experimental foi inteiramente casualizado com quatro tratamentos (quatro níveis de inclusão de enzimas, 0,0; 0,05; 0,10; e 0,15 %), três repetições (no tempo) e 10 peixes por unidade experimental. Foram utilizados 40 juvenis de tambaqui, com peso médio de 155,0 ± 0,49 g, distribuídos em quatro tanques de alimentação de 500 l, recebendo refeições à vontade das 8 às 12h, a cada hora. Em seguida os animais foram transferidos para coletores de fezes (200 l), onde permaneceram até às 18h, sendo a coleta de dejetos realizada a cada hora. A determinação dos CDa foi realizada pelo método indireto, sendo utilizado como indicador externo 0,5% de óxido de cromo-III (Cr2O3) incorporado à ração. Os resultados demonstraram que a suplementação das dietas com enzimas exógenas para juvenis de tambaqui aumenta a digestibilidade aparente dos nutrientes e energia bruta, no nível de inclusão de 0,05% (P<0,05%).

Clivagem e caracterização de frutalina recombinante produzida em Escherichia coli

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Expressão e caracterização de uma lectina recombinante de interesse biomédico

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Combined bioremediation and enzyme production by Aspergillus sp. in olive mill and winery wastewaters

Olive mill wastewaters (OMW) and vinasses (VS) are effluents produced respectively by olive mills and wineries, both sectors are of great economic importance in Mediterranean countries. These effluents cause a large environmental impact, when not properly processed, due to their high concentration of phenolic compounds, COD and colour. OMW may be treated by biological processes but, in this case, a dilution is necessary, increasing water consumption. The approach here in proposed consists on the bioremediation of OMW and VS by filamentous fungi. In a screening stage, three fungi (Aspergillus ibericus, Aspergillus uvarum, Aspergillus niger) were selected to bioremediate undiluted OMW, two-fold diluted OMW supplemented with nutrients, and a mixture of OMW and VS in the proportion 1:1 (v/v). Higher reductions of phenolic compounds, colour and COD were achieved mixing both residues; with A. uvarum providing the best results. In addition, the production of enzymes was also evaluated during this bioremediation process, detecting in all cases lipolytic, proteolytic and tannase activities. A. ibericus, A. uvarum and A. niger achieved the highest value of lipase (1253.7 ± 161.2 U/L), protease (3700 ± 124.3 U/L) and tannase (284.4 ± 12.1 U/L) activities, respectively. Consequently, this process is an interesting alternative to traditional processes to manage these residues, providing simultaneously high economic products, which can be employed in the same industries.