Disaggregases, molecular chaperones that resolubilize protein aggregates

The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of pro872012731292FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORSEM INFORMAÇÃOSEM INFORMAÇÃOSEM INFORMAÇÃOO processo de enovelamento é um evento importante para o tempo de vida de uma proteína, uma vez que é essencial para a função adequada da proteína e, por conseguinte, para a fisiologia celular. Enovelamento inadequado, ou mau enovelamento, pode não só conWe thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and fellowship

Conformational and functional characterization of AAA domain proteins through the investigation of human ANKCLP and Rvbs from Leishmania major and Saccharomyces cerevisiae

Orientador: Carlos Henrique Inacio RamosTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A família de proteínas AAA+ são enzimas ATPases associadas a inúmeras atividades celulares. Caracterizadas principalmente pela presença do domínio de ligação e hidrólise de nucleotídeos, conhecido como NBD (Nucleotide Binding Domain), as proteínas AAA+ possuem funções que compreendem desde a replicação do DNA até a degradação de proteínas. Dessa forma, esta tese descreve os resultados obtidos com algumas proteínas com domínio AAA+: ANKCLP humana e as Rvbs 1 e 2 de Leishmania major. A proteína ANKCLP humana possui um domínio de ligação e hidrólise de ATP, que é semelhante às chaperonas desagregases de outros organismos. Com isso, utilizando o modelo de estudo em leveduras, nossos resultados mostraram que a proteína ANKCLP humana não auxiliou na sobrevivência em células com o gene deletado da Hsp104, chaperona desagregase bem caracterizada em leveduras. Além disso, a ANKCLP não foi capaz de remodelar os agregados amilóides nas células de leveduras que continham fatores para formação de príons. Devido a semelhança com a ANKCLP, nos interessamos pelas características da proteína Hsp78, chaperona desagregase de mitocôndria de leveduras. Sendo assim, produzimos uma mini-revisão de literatura sobre esta proteína e uma visão crítica sobre a proteína ANKCLP ser uma chaperona desagregase em animais. Também neste trabalho foram estudadas as proteínas Rvbs que também possuem um domínio NBD e foram pela primeira vez descritas no organismo Leishmania major, causador da leishmaniose em mamíferos. De fato, estas helicases apesar de serem bem conhecidas em leveduras e humanos, são bem menos estudadas quando se trata de protozoários. Com isso, a Rvb1 e Rvb2 de L. major foram purificadas com sucesso, sendo que a LmRvb1 foi capaz de formar um pentâmero em solução, mas não possui atividade ATPásica in vitro. Já a LmRvb2 forma um grande oligômero e possui atividade ATPásica in vitro. A co-expressão destas proteínas demonstrou a presença de pelo menos um dímero em solução. Experimentos de estabilidade e fluorescência indicaram que estas proteínas interagem nestas condições e este dímero possui atividade ATPásica in vitro, assim como a LmRvb2. Por fim, com este trabalho foi possível compreender parte da função e conformação de algumas proteínas da família AAA+ e esperamos contribuir para o maior entendimento destas proteínas nos diferentes organismosAbstract: AAA+ proteins family are ATPase enzymes associated with diverse cellular activities. Mainly characterized by the presence of the nucleotide binding domain, known as NBD (Nucleotide Binding Domain), the AAA proteins can act of a ranging of functions from DNA replication to protein degradation. Thus, this thesis describes the results obtained with some AAA+ proteins: human ANKCLP and Rvbs 1 and 2 from Leishmania major. The human ANKCLP protein has a NBD domain which is very similar to the NBD domais from chaperone disaggregases. Therefore, using the yeast model, we showed that the ANKCLP protein didn¿t complement the yeast cell survival in the absence of Hsp104, chaperone disaggregate with two AAA+ domains and well characterized in yeast. In addition, the ANKCLP was not able to remodel amyloid aggregates in yeast cells which contain prion formation factors. Due to the similarity with ANKCLP, we were interested in the characteristics of the Hsp78 protein, chaperone disaggregate from yeast mitochondria. Thus, we have produced a mini-review on this protein and a critical view about the ANKCLP protein as a hypothetical chaperone disaggregase in animals. Also, is this work we studied the proteins Rvbs which also have one NBD domain and, in the first time, they have been described from Leishmania major, which causes leishmaniasis in mammals. Indeed, although these proteins are well described from human and yeast, they are not well studied when it comes from protozoa. Therefore, the Rvb1 and Rvb2 were successfully purified, and LmRvb1 was able to form a pentamer in solution, but did not have ATPase activity in vitro. LmRvb2 forms a large oligomer and has ATPase activity in vitro. The co-expression of Rvb1 and Rvb2 formed at least a dimer in solution. Stability and fluorescence experiments indicated that these proteins interact under these conditions and this dimer has ATPase activity in vitro, as well as an LmRvb2. Finally, with this work we hope to contribute to greater understanding of these AAA+ proteinsDoutoradoBioquimicaDoutora em Biologia Funcional e Molecular2013/10939-2FAPES

Hsp78 (78 kDa Heat Shock Protein), a Representative AAA Family Member Found in the Mitochondrial Matrix of Saccharomyces cerevisiae

ATPases associated with diverse cellular activities (AAA+) form a superfamily of proteins involved in a variety of functions and are characterized by the presence of an ATPase module containing two conserved motifs known as Walker A and Walker B. ClpB and Hsp104, chaperones that have disaggregase activities, are members of a subset of this superfamily, known as the AAA family, and are characterized by the presence of a second highly conserved motif, known as the second region of homology (SRH). Hsp104 and its homolog Hsp78 (78 kDa heat shock protein) are representatives of the Clp family in yeast. The structure and function of Hsp78 is reviewed and the possible existence of other homologs in metazoans is discussed

Characterization of the Hsp100 disaggregase from sugarcane (SHsp101) for chaperone like activity in a yeast system

FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe Hsp104/ClpB protein subfamily is unique in its ability to reactivate protein aggregates, which are implicated in several human and animal diseases. However, when compared to the unicellular yeast Hsp104 and bacterial ClpB disaggregases, the multicellu26478487FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2012/50161-8We thank Claudio Masuda (UFRJ) for gifting the BY4741 yeast strain and isogenic Hsp104 knockout, and Doug Cyr (University of North Carolina) for the Rnq-mRFP yeast expression plasmid, and the W303 [RNQ+] yeast strai

Aluminum oxide nanoparticles affect the cell wall structure and lignin composition slightly altering the soybean growth

Aluminum oxide (Al2O3) nanoparticles (NPs) are among the nanoparticles most used industrially, but their impacts on living organisms are widely unknown. We evaluated the effects of 50-1000 mg L-1 Al2O3 NPs on the growth, metabolism of lignin and its monomeric composition in soybean plants. Al2O3 NPs did not affect the length of roots and stems. However, at the microscopic level, Al2O3 NPs altered the root surface inducing the formation of cracks near to root apexes and damage to the root cap. The results suggest that Al2O3 NPs were internalized and accumulated into the cytosol and cell wall of roots, probably interacting with organelles such as mitochondria. At the metabolic level, Al2O3 NPs increased soluble and cell wall-bound peroxidase activities in roots and stems but reduced phenylalanine ammonia-lyase activity in stems. Increased lignin contents were also detected in roots and stems. The Al2O3 NPs increased the p-hydroxyphenyl monomer levels in stems but reduced them in roots. The total phenolic content increased in roots and stems; cell wall-esterified p-coumaric and ferulic acids increased in roots, while the content of p-coumaric acid decreased in stems. In roots, the content of ionic aluminum (Al+3) was extremely low, corresponding to 0.0000252% of the aluminum applied in the nano-particulate form. This finding suggests that all adverse effects observed were due to the Al2O3 NPs only. Altogether, these findings suggest that the structure and properties of the soybean cell wall were altered by the Al2O3 NPs, probably to reduce its uptake and phytotoxicity

Biosynthesis and metabolic actions of simple phenolic acids in plants

The diversity of secondary compounds in the plant kingdom is huge. About 200,000 compounds are known, which are grouped into amines, non-protein amino acids, peptides, alkaloids, glucosinolates, cyanogenic glucosides, organic acids, terpenoids, quinones, polyacetylenes, and phenolics. The group of phenolic compounds consists of polyphenols, oligophenols and monophenols or simple phenolic compounds such as benzoic and cinnamic acids and their hydroxylated derivatives. Among the thousands of compounds present in ecological interactions, simple phenolic acids are the most abundant in soils, and many are described as allelochemicals. Given the physiological and biochemical importance of these compounds, we review their biosynthesis and metabolic actions in plants