No description available.

Inibidores de proteases - moléculas capazes de inibir a atividade catalítica de enzimas proteolíticas - compreendem uma das mais abundantes classes de proteínas em plantas. Nas sementes, as funções destas moléculas estão relacionadas com o controle de proteínas endógenas e com mecanismo de defesa contra insetos fitófagos. Este trabalho teve como objetivo isolar, purificar, caracterizar estruturalmente e testar algumas aplicações biotecnológicas de um inibidor de serinoprotease das sementes de Cassia leptophylla. Após extração salina, a partir de cotilédones de sementes secas, o inibidor foi purificado por meio de cromatografia de filtração molecular sobre coluna Superdex75 e cromatografia de troca iônica em uma coluna Mono-Q. A proteína purificada apresentou PI de aproximadamente 4,5 e massa molecular estimado por eletroforese (SDS-PAGE) de 20kDa, sendo constituída por duas cadeias polipeptídicas de l6kDa e 4kDa. A seqüência N-terminal da cadeia de 4kDA (ATEDEKKDLGISIDDCCNRRLVVK) revelou similaridade com outros inibidores de serinoproteases tipo Kunitz isolados de Adenanthera pavonina, Prosopsis juliflora e Acacia confusa; o inibidor em estudo foi denominado CLTI (Cassia 1eptophyZZa trypsin inhibitor). A investigação da seqüência N-terminal da cadeia de 16 kDa, por meio da degradação de Edman automatizada, não deu sinal positivo, indicando provável bloqueio do primeiro resíduo de aminoácido. O espectro de dicroísmo circular (CD) do inibidor revelou que os componentes de estrutura secundária são constituídos predominantemente de folhas-&#946, voltas e estruturas não ordenadas. O espectro de emissão de fluorescência deste inibidor apresentou máximo de emissão em 226 nm, típico de proteínas com os resíduos de triptofano protegidos do solvente. CLTI quando submetido a pHs extremos (ácidos e básicos) revelou alterações de estrutura secundária e foi estável até cerca de 55&#176C (temperatura de transição 59&#176C), ambos investigados por CD e emissão de fluorescência. Ensaios de atividade biológica in vitro revelaram que CLTI inibiu a coagulação de plasma humano citratado, a ação das enzimas tripsina (KI 1,92&#956M), quimotripsina (KI 14,5&#956M), e calicreína plasmática humana (KI 1,5&#956M), mas não apresentou inibição sobre elastase porcina. CLTI formou um complexo estável com a tripsina demonstrado pela eluição do mesmo em coluna de filtração molecular. Este inibidor também mostrou atividade fungicida sobre as cepas de Fusarium monifome e Fusarium gramineanim e atividade fungistática sobre as cepas de Colletotrichum sp. F37 e Colletotrichum sp. P10. / Proteinase inhibitors are molecules able to inhibit the catalytic activity of proteolytic enzymes. They are the most abundant class of plant proteins. The function of these molecules in seeds is related to the control of endogenous proteins and defense mechanisms against phytopathogens. The objectives of this work are the isolation and structural characterization of a serine proteinase inhibitor from seeds of Cassza leptophylla. Besides, some biotechnological properties of this inhibitor were tested. After saline extraction from dry seed cotyledons, the inhibitor was purified by gel filtration chromatography on a Superdex75 column and ion exchange chromatography on a Mono-Q column. The purified protein presents a pI of approximately pH 4,5 and 20 kDa molecular mass estimated by electrophoresis (SDS-PAGE). This protein is constituted by two polypeptide chains, one of l6kDa and other of 4kDa. The N-terminal sequence of 4kDa peptide (ATEDEKKDLGISIDDCCNRRLVVK) reveals homology with other Kunitz type serine proteases inhibitors isolated from Adenanthera pavonina, Prosopsis juliflora and Acacia confisa. The studied inhibitor was named CLTI (Cassza leptophylla trypsin inhibitor). N-terminal sequencing of the l6kDa chain, using Edman degradation, did not show positive signal probably indicating blocked amino acid residues. The circular dichroism spectrum (CD) of this inhibitor reveals that the components of the secondary structure are constituted predominantly by sheets-&#946 and unordered structures. The fluorescence emission spectrum of this inhibitor presents the maximum emission at 226nm typical of proteins with tryptophan residues solvent inaccessible. CLTI secondary structure was revealed to change at extremely acid and alkaline pHs. It remained stable at temperatures up to 55 &#176C (transition temperature of this protein is 59&#176C). All these data were investigated by CD and fluorescence emission. Tests of biological activity in vitro revealed that CLTI inhibits blood clotting of citrated human plasm. Tests also showed inhibitory activity against trypsin (Ki 1,92&#956M), chymotrypsin (Ki 14,5&#956M) and human plasm kallikrein (Ki 1,5&#956M), but did not present inhibition against porcine pancreatic elastase. CLTI showed to form a stable complex with trypsin when eluted in gel filtration chromatography. This inhibitor also showed fungicide activity against the strains of Fusarium moniforme and Fusarium graminearum, and fungistatic activity against Colletotrichum sp. F37 and Colletotrichum sp. P10 strains.

Inibidores de proteases - moléculas capazes de inibir a atividade catalítica de enzimas proteolíticas - compreendem uma das mais abundantes classes de proteínas em plantas. Nas sementes, as funções destas moléculas estão relacionadas com o controle de proteínas endógenas e com mecanismo de defesa contra insetos fitófagos. Este trabalho teve como objetivo isolar, purificar, caracterizar estruturalmente e testar algumas aplicações biotecnológicas de um inibidor de serinoprotease das sementes de Cassia leptophylla. Após extração salina, a partir de cotilédones de sementes secas, o inibidor foi purificado por meio de cromatografia de filtração molecular sobre coluna Superdex75 e cromatografia de troca iônica em uma coluna Mono-Q. A proteína purificada apresentou PI de aproximadamente 4,5 e massa molecular estimado por eletroforese (SDS-PAGE) de 20kDa, sendo constituída por duas cadeias polipeptídicas de l6kDa e 4kDa. A seqüência N-terminal da cadeia de 4kDA (ATEDEKKDLGISIDDCCNRRLVVK) revelou similaridade com outros inibidores de serinoproteases tipo Kunitz isolados de Adenanthera pavonina, Prosopsis juliflora e Acacia confusa; o inibidor em estudo foi denominado CLTI (Cassia 1eptophyZZa trypsin inhibitor). A investigação da seqüência N-terminal da cadeia de 16 kDa, por meio da degradação de Edman automatizada, não deu sinal positivo, indicando provável bloqueio do primeiro resíduo de aminoácido. O espectro de dicroísmo circular (CD) do inibidor revelou que os componentes de estrutura secundária são constituídos predominantemente de folhas-&#946, voltas e estruturas não ordenadas. O espectro de emissão de fluorescência deste inibidor apresentou máximo de emissão em 226 nm, típico de proteínas com os resíduos de triptofano protegidos do solvente. CLTI quando submetido a pHs extremos (ácidos e básicos) revelou alterações de estrutura secundária e foi estável até cerca de 55&#176C (temperatura de transição 59&#176C), ambos investigados por CD e emissão de fluorescência. Ensaios de atividade biológica in vitro revelaram que CLTI inibiu a coagulação de plasma humano citratado, a ação das enzimas tripsina (KI 1,92&#956M), quimotripsina (KI 14,5&#956M), e calicreína plasmática humana (KI 1,5&#956M), mas não apresentou inibição sobre elastase porcina. CLTI formou um complexo estável com a tripsina demonstrado pela eluição do mesmo em coluna de filtração molecular. Este inibidor também mostrou atividade fungicida sobre as cepas de Fusarium monifome e Fusarium gramineanim e atividade fungistática sobre as cepas de Colletotrichum sp. F37 e Colletotrichum sp. P10. / Proteinase inhibitors are molecules able to inhibit the catalytic activity of proteolytic enzymes. They are the most abundant class of plant proteins. The function of these molecules in seeds is related to the control of endogenous proteins and defense mechanisms against phytopathogens. The objectives of this work are the isolation and structural characterization of a serine proteinase inhibitor from seeds of Cassza leptophylla. Besides, some biotechnological properties of this inhibitor were tested. After saline extraction from dry seed cotyledons, the inhibitor was purified by gel filtration chromatography on a Superdex75 column and ion exchange chromatography on a Mono-Q column. The purified protein presents a pI of approximately pH 4,5 and 20 kDa molecular mass estimated by electrophoresis (SDS-PAGE). This protein is constituted by two polypeptide chains, one of l6kDa and other of 4kDa. The N-terminal sequence of 4kDa peptide (ATEDEKKDLGISIDDCCNRRLVVK) reveals homology with other Kunitz type serine proteases inhibitors isolated from Adenanthera pavonina, Prosopsis juliflora and Acacia confisa. The studied inhibitor was named CLTI (Cassza leptophylla trypsin inhibitor). N-terminal sequencing of the l6kDa chain, using Edman degradation, did not show positive signal probably indicating blocked amino acid residues. The circular dichroism spectrum (CD) of this inhibitor reveals that the components of the secondary structure are constituted predominantly by sheets-&#946 and unordered structures. The fluorescence emission spectrum of this inhibitor presents the maximum emission at 226nm typical of proteins with tryptophan residues solvent inaccessible. CLTI secondary structure was revealed to change at extremely acid and alkaline pHs. It remained stable at temperatures up to 55 &#176C (transition temperature of this protein is 59&#176C). All these data were investigated by CD and fluorescence emission. Tests of biological activity in vitro revealed that CLTI inhibits blood clotting of citrated human plasm. Tests also showed inhibitory activity against trypsin (Ki 1,92&#956M), chymotrypsin (Ki 14,5&#956M) and human plasm kallikrein (Ki 1,5&#956M), but did not present inhibition against porcine pancreatic elastase. CLTI showed to form a stable complex with trypsin when eluted in gel filtration chromatography. This inhibitor also showed fungicide activity against the strains of Fusarium moniforme and Fusarium graminearum, and fungistatic activity against Colletotrichum sp. F37 and Colletotrichum sp. P10 strains.

Capsid of mouse polyomavirus (MPyV) is composed from three structural proteins: major structural protein VP1 and minor structural proteins VP2 and VP3. Posttranslational modifications may affect functions of proteins. This work deals with acetylation of MPyV structural proteins and its impact on the viral replication cycle. First part of the thesis is focused on acetylation of VP1. We showed that the VP1 protein is acetylated in viral particles and that interaction of VP1 with minor proteins supports VP1 acetylation. Further, we showed that cytoplasmatic deacetylase, histone deacetylase 6 (HDAC6), is important for virus infectivity. Overexpression of HDAC6 decreased MPyV infectivity, also decreased infectivity was exhibited by virus isolated from HDAC6 knock out cells. In addition, VP1 protein of virus from HDAC6 knock out cells was more acetylated in comparison with virus from parental cell line. These data suggest that VP1 is substrate for HDAC6. Second part of the thesis is focused on the characterization of N-terminal acetylation of VP3 minor structural protein. It has been previously shown that VP3 protein is N-terminally acetylated and MyPV with mutated (unacetylated) form of VP3 protein is non-infectious. The main aim of this part is to prove the hypothesis that N-terminal acetylation is...

No description available.

Background:  The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective:  The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods:  The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.

This thesis evaluates the organization of the structural proteins, Human Gamma D crystallin and Collagen type II, into higher-order structures using light scattering. Specifically, it evaluates the natures of incipient aggregation in Human Gamma D crystallin and the nature of its interactions with CAPEGn, an electrostatic blocker. Additionally, this thesis evaluates whether Collagen type II growth kinetics follows Classical nucleation theory.

Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.

No description available.

Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.