ISG15 is an interferon-induced and anti-viral ubiquitin-like protein (Ubl). Ube1L, UbcH8, and Herc5 have been identified as the E1-E2-E3 enzymes for ISG15 conjugation, and, like ISG15, their expression is induced by type I interferons. Although Herc5 is the major E3 for ISG15, over 300 proteins have been identified as ISG15 target proteins in interferon-stimulated cells. In this work, I address two aspects of the human ISG15 conjugation system: 1) the specificity of the Ube1L-UbcH8 interaction and 2), the basis of substrate recognition by Herc5. Regarding the selection of UbcH8 by Ube1L, my experiments show that although UbcH8 had been reported to function as an E2 for both Ub and ISG15, UbcH8 is preferentially activated by Ube1L compared to Ube1 (E1[superscript Ub]). The basis of this preference is a result of specific interactions between the ubiquitin-fold domain (UFD) of Ube1L and the amino-terminal [alpha]1 helix and [beta]1 [beta]2 region within UbcH8. Examination of the interferon-induced and transfected expression levels of UbcH8, combined with the kinetic constants, suggest that UbcH8 is unlikely to function as a Ub E2 in most cell lines. In examining the selection of target proteins by Herc5, I show that the range of substrates extends far beyond the proteins identified in proteomics studies and includes many exogenously expressed foreign proteins. Furthermore, I show that ISG15 conjugation is restricted to newly synthesized pools of proteins and Herc5 is associated with polyribosomes. I propose a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a co-translational manner and suggest that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, I show that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses. These discoveries have greatly increased our understanding of the mechanism of ISG15 pathway and provide a framework for establishing an in vitro ISG15 conjugation system and further examination of the anti-viral function of ISG15. / text

Two groups of growth regulators were described in Drosophila imaginal disc cell culture Cl.8+. Imaginal disc growth factors (IDGFs) belonging to chitinase-like protein family of carbohydrate binding proteins and Adenosine deaminase-related growth factors (ADGFs), which are active adenosine deaminases influencing homeostasis of key cellular metabolite adenosine. The functions of two of the IDGFs, as well as the effects of extracellular adenosine and its receptor were studied primarily in in vitro cell culture. Our results supported their roles in the regulation of cell survival and energy homeostasis especially in imaginal disc cells. Both the IDGFs and adenosine also play important roles in organismal responses to stress and infection and may interact in vivo.

Orientadores: Gonçalo Amarante Guimarães Pereira, Odalys Garcia Cabrera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T12:21:46Z (GMT). No. of bitstreams: 1 Zaparoli_GustavoHenriqueAlcala_D.pdf: 22559337 bytes, checksum: f8c948505b53f1b1be80dd4146ce6a2b (MD5) Previous issue date: 2012 / Resumo: Moniliophthora perniciosa e Moniliophthora roreri são dois fungos basidiomicetos hemibiotróficos que causam as doenças Vassoura-de-Bruxa e Monilíase, respectivamente, em cacaueiros da América Central e do Sul. M. perniciosa ataca meristemas de ramos e frutos em desenvolvimento enquanto M. roreri é capaz de infectar unicamente os frutos, sendo ambas as doenças devastadoras nas plantações afetadas. Nos genomas de M. perniciosa e M. roreri foram encontrados genes que codificam pequenas proteínas secretadas, descritas como efetores, com potencial importância nas interações patógenos- planta, entre eles estão membros das famílias de proteínas NEP-1 like proteins (NLPs) e Ceratoplataninas (CP). As NLPs causam necrose após contato com as células planta e em M. perniciosa identificamos uma cópia (MpNEP2) expressa simultaneamente com os sintomas de necrose da doença. MpNEP2 teve sua estrutura resolvida por cristalografia e apresenta semelhanças com citotoxinas. Identificamos aminoácidos essenciais à atividade de necrose de MpNEP2, e analisamos sua possível dependência de íons. As CPs são proteínas descritas como elicitores de resposta de defesa em plantas, e nas espécies de Moniliophthora parecem ter uma função relacionada à proteção estrutural durante a doença. Análises de expressão mostraram grande diferença entre as cópias identificadas em M. perniciosa. Através da obtenção e análises das estruturas cristalográficas de quatro cópias de MpCPs sugerimos possíveis funções a essa família durante ambas as doenças. O objetivo principal deste estudo foi a compreensão dos mecanismos de ação destas duas classes de elicitores e sua relação no desenvolvimento de cada uma das doenças / Abstract: Moniliophthora perniciosa and Moniliophthora roreri are two hemibiotrophic fungi that cause Witches' Broom and Frosty Pod Rod Diseases, respectively, in cocoa trees of Central and South America. ...Note: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Philosophiae Doctor - PhD / Global climate change has resulted in altered rainfall patterns, causing annual losses in maize crop yield due to water deficit stress. Therefore, it is important to produce maize cultivars which are more drought-tolerant. This not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of water deficit stress. The RD22-like protein subfamily is expressed in response to water deficit stress. Members of the RD22-like subfamily include AtRD22, GmRd22 and BnBDC1 which have been identified in Arabidopsis thaliana, Glycine max and Brassica napus respectively. This study aims at characterising two putative maize RD22-like proteins (designated ZmRd22A and ZmRd22B) by identifying sequence/domain features shared with characterised RD22-like proteins. Semi-quantitative and quantitative PCR techniques were used to examine the spatial and temporal expression patterns of the two putative maize Rd22-like proteins in response to, water deficit stress and exogenously applied abscisic acid in the roots and 2nd youngest leaves of maize seedlings. Using an in silico approach, sequence homology of the two putative maize Rd22- like proteins with AtRD22, GmRD22 and BnBDC1 has been analysed. Online bioinformatic tools were used to compare the characteristics of these Rd22-like proteins with those of the two maize proteins. It was shown that the putative maize RD22-like proteins share domain organisation with the characterised proteins, these common features include a N-terminal hydrophobic signal peptide, followed by a region with a conserved amino acid sequence, a region containing several TxV (x is any amino acid) repeat units and a C-terminal BURP domain-containing the conserved X₅-CH-X₁₀-CH-X₂₃-₂₇-CH-X₂₃-₂₆-CH-X₈-W motif. The putative maize Rd22-like protein appears to be localized in the apoplast, similarly to AtRD22, GmRD22 and BnBDC1. Analysis of the gene's promotor regions reveals cis-acting elements suggestive of induction of gene expression by water deficit stress and abscisic acid (ABA). Semi-quantitative and quantitative real time PCR analysis of the putative maize RD22-like gene revealed that the genes are not expressed in the roots. Exposure to water deficit stress resulted in an increase of ZmRD22A transcript accumulation in the 2nd youngest leaves of maize seedlings. ZmRD22A was shown to be non-responsive to exogenous ABA application. ZmRD22B was highly responsive to exogenous ABA application and responded to water deficit stress to a lesser degree. Transcript accumulation studies in three regions of the 2nd youngest leaves in response to water deficit stress showed that ZmRd22A transcripts accumulate mainly at the base and tips of the leaves. A restricted increase in ZmRD22A transcript accumulation in the middle of the leaves was observed. ZmRD22B showed a similar, but weaker transcript accumulation pattern in response to water deficit stress. However, ZmRD22B showed increased transcript accumulation in the middle region of the leaves. In response to exogenous ABA application, ZmRd22B exhibited high transcript accumulation at the base of the 2nd youngest leaves, with the middle showing higher transcript accumulation than the tip of the leaves. It was concluded that ZmRD22A and ZmRD22B share the domain organisation of characterised RD22-like proteins as well as being responsive to water deficit stress, although only ZmRD22B was shown to be responsive to exogenous ABA application. / National Research Foundation (NRF)

Group A Streptococcus (GAS) is a human specific bacterial pathogen which causes a variety of diseases and is responsible for significant mortality worldwide. GAS are known to interact with the immune system, notably by binding host serum proteins to the bacterial surface.Many of these binding functions are attributed to the GAS M protein, the archetypal GAS virulence factor, the substrate for GAS typing and the leading GAS vaccine candidate. The vast diversity of GAS strains has however made vaccine development challenging. We investigated the potential for cross-protective immunity within closely related strains in a clinical setting in Fiji. This study has shown that immunity to GAS skin infection was broader than previously believed and included some level of cross-opsonisation betweenGAS strains. The level of such cross-opsonisation was, however, variable among GAS lineages. We have also shown that the immunity to conserved M antigens was quite variable. These results inspired us to investigate other suitable vaccine antigens. We hypothesised that in cases where the M protein was less immunodominant, perhaps closely related M-like proteins played an important role in immunity. We therefore began global characterisation ofthe two major M-like proteins, called Mrp and Enn. A representative worldwide genomic collection including more than 2000 isolates originating from multiple continents and various clinical manifestations has been established collaboratively. We focused our analyses on theMga regulon which encodes M and M-like proteins. We found that mrp and enn genes were present in 85% of genomes suggestive of their importance to GAS survival and spread. We developed molecular definitions of the different genes families, clarifying nomenclature forthe worldwide reference laboratory at the American CDC. We established and validated an updated, more specific typing protocol for GAS which will reduce future misclassifications. We have also analysed the genetic linkage between M and M-like protein alleles and developed clusters of closely related protein sequences. By characterising this complex and variable genetic region, we provide a framework for future functional investigations. Finally, we began functional characterisation of Enn proteins by investigating the differential capacity of Enn proteins to bind to C4BP, an inhibitor of complement. Altogether our results suggest M-like proteins play an important role in GAS virulence and should not be neglected. These data support further functional analyses to better understand the contribution of M-like proteins to GAS infection. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

No description available.

Myxococcus xanthus Dif proteins are chemotaxis homologues that regulate exopolysaccharide (EPS) biogenesis. Previous genetic studies suggested that Dif protein might interact with one another as do the chemotaxis proteins in enterics. The interactions among Dif proteins were since investigated with the yeast two-hybrid (Y2H) system. The results indicate that DifC interacts with both DifA and DifE. Using a modified Y2H system, DifC was shown to be able to bring DifA and DifE into a protein complex. Further Y2H experiments demonstrated that the different conserved domains of DifE likely function as their counterparts of CheA-type kinases because the putative P2 domain of DifE interacts with DifD, P5 with DifC and the dimerization domain P3 with itself. Similarly, DifA can interact with itself through its C-terminal region. In addition, DifG was found to interact with the CheY homologue DifD. These findings support the notion that Dif proteins constitute a unique chemotaxis-like signal transduction pathway in M. xanthus. In addition, KapB, a TPR (Tetratricopeptide repeats) protein, was identified as an interacting partner of DifE byY2H library screening. Further analysis demonstrated that the N-terminal half of KapB interacted with the putative P2 domain of DifE. KapB had been previously reported to interact with several Serine/Threonine (Ser/Thr) kinase pathways including the Pkn4-Pfk pathway. This pathway is implicated in glycogen metabolism in M. xanthus by a previous report. In this study, kapB as well as pfkn deletion mutants were found to overproduce EPS. It was also found that the Dif pathway is involved in glycogen metabolism because the glycogen level is altered in dif mutants. These results indicate EPS biogenesis and glycogen metabolism may be coordinately regulated. This coordination of the Dif-regulated EPS production and the Pkn4-regulated glycogen metabolism appears to involve KapB. This is the first example of a TPR protein mediating the interplays of a histidine kinase pathway and a Ser/Thr kinase pathway. / Master of Science

Ice structuring proteins (ISPs) naturally exist in many foods consumed as part of the human diet including plants or fish. ISPs from winter wheat grass have gained interest in the pharmaceutical and food industries as a non-toxic, natural and cost-effective product, which is easy to prepare as a crude extract. However, they have not been purified reproducibly and studied in detail to elucidate their structures, mechanism of actions and difference(s). ISPs from the apoplast region of cold acclimated winter wheat leaves were extracted through vacuum infiltration and purified using heat and ethanol precipitations, size exclusion and anionic exchange fast protein liquid chromatography techniques. The ISPs showed both significant inhibition of ice growth and thermal hysteresis activities. The non-acclimated apoplastic extracts from winter wheat leaves contained similar proteins without any abovementioned activities. The ISPs contained disulfide bridges, similar to thaumatin-like proteins (TLPs) and partially similar to ISPs from winter rye leaves and carrot. ISPs remained active after thermal treatment (i.e., pasteurization conditions) and over a wide range of pH (3-12). There are very few quantitative assays to measure the activity of antifreeze proteins (AFPs, or Ice Structuring Proteins, ISPs), which often suffer from various inaccuracies and inconsistencies. Some methods rely only on unassisted visual assessment. When microscopy is used to measure ice crystal size, it is critical that standardized procedures be adopted, especially when image analysis software is used to quantify sizes. Differential Scanning Calorimetry (DSC) has been used to measure the thermal hysteresis activity (TH) of AFPs. In this study, DSC was used isothermally to measure enthalpic changes associated with structural rearrangements as a function of time. Differences in slopes of thermograms between winter wheat ISP or AFP type I containing samples, and those without ISP or AFP type I were demonstrated. ISP or AFP type I containing samples had much higher slopes compared to those without ISP or AFP type I. Samples with higher concentration of ISP or AFP type I showed higher slope values. The proteinaceous activity of ISPs or AFP type I was confirmed by demonstrating changes in samples with and without proteases. A proposed mechanism of this method is discussed.

No description available.

No description available.