Search

Background : Glycogen storage disease type II or Pompe disease is a rare inherited metabolic. Pompe disease is caused by mutations in the gene that encodes the lysosomal hydrolase acid a-glucosidase (GAA). GAA deficiency results in lysosomal glycogen accumulation and cellular dysfunction in multiple tissues, particularly skeletal and cardiac muscle . Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA; Myozyme, Genzyme Corporation, Cambridge, MA) is the only approved treatment for Pompe disease. Recently, it has been demonstrated that pharmacological chaperones, small molecules that specifically bind and stabilize target proteins, can also enhance the trafficking and cellular activity of a number of different wild-type and mutant forms of lysosomal hydrolases. A general strategy for lysosomal storage disorder is also to search for small pharmacological chaperones that are able to bind the active site of mutant enzymes and to facilitate the folding and transport process. These molecules have the ability to improve the trafficking of the protein between the endoplasmic reticulum (ER) and the lysosome. Various inhibitors derived from deoxynojirimycin (DNJ) have been evaluated as pharmacological chaperones, in different lysosomal storage disorders . Methods : We examined the binding affinity of pharmacological chaperones such as 1-deoxynojirimycin (DNJ) and N-acetylcysteine (NAC) to the rhGAA, Myozyme by Isothermal Titration Calorimetry. We studied thermal stability of the enzyme in the absence and presence of ligands at neutral and acidic pH by Differential Scanning Calorimetry Results : ITC results allowed a complete thermodynamic characterization of the interaction between the studied small ligand and the enzyme while the DSC results gave important information on the enzyme conformational stability as a function of pH.

Carbohydrates play an important role in a variety of biological and industrial processes. The elucidation of the structure/function relationship of glycoside hydrolases (GHs) can help us to understand their implication in various biological phenomena and to exploit their catalytic features, i. e. in pharmacological therapies or to optimize industrial processes. On the basis of these observations, this thesis work is directed to the study of GHs structure/function relationship and, besides the general introduction illustrated in chapter 1, it can be subdivided in two principal experimental sections (chapter 2 and 3). In chapter 2, the research plan was focused on the identification and biochemical characterization of novel glycosidases activities in the hyperthermophilic crenarchaeon Sulfolobus solfataricus. This study allowed to identify the first N-acetylglucosaminidase in this living domain, to increase our knowledge on the phylogeny of this class of enzymes, and to achieve additional data concerning their structural and functional features. Chapter 3 reports the structure/function study of the human lysosomal α-glucosidase, shading light on the factors responsible of its stabilization. The informations gained are exploited to identify novel pharmacological chaperones for the therapy of the lysosomal storage disorder known as Pompe disease.

Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level., Pompe disease is caused by mutations in lysosomal acid α-glucosidase (GAA) and patients are being treated with recombinant human α-glucosidase (rhGAA). Here the authors present the crystal structures of rhGAA and its complexes with inhibitors and a pharmacological chaperone, which is important for drug development.

The second generation bioethanol represents a main challenge in global efforts to utilize renewable resources rather than fossil fuels. However, the close association of cellulose and hemicelluloses to lignin in the plant cell wall makes it difficult to degrade lignocellulose into fermentable sugars. Consequently, pretreatments are necessary to make the polysaccharides more accessible to the enzymes, but the high temperature and extreme pH conditions required give rise to problems when using conventional enzymes in the saccharification step (Galbe and Zacchi 2002). Microorganisms thriving in habitats characterized by harsh conditions, and the enzymes derived therein, represent a helpful tool in the development of bioethanol production processes. In fact, they allow bioconversions at non-conventional conditions under which common biocatalysts are denatured. The use of high operational temperatures allows energy savings by reducing the cooling cost after high temperature pretreatments, and, in ethanol production, thermophilic conditions permit ethanol evaporation allowing harvest during fermentation.