Characterization of human lysyl hydroxylase LH2a, a collagen modifying enzyme.

Collagen is the most abundant protein in the human body: it represents the 30% of the total protein mass in mammals. Humans contain five principal collagen types (I-V), according to their localization in the different tissues. The collagen maturation pathway is one of the most complex and intricate biosynthetic process. After protein translation, the procollagen peptides undergo a plethora of posttranslational modifications, including proline hydroxylation, lysine hydroxylation, and N- and O-glycosylations. The mature collagen molecules are finally secreted in the extracellular matrix (ECM), where they mainly play a structural role: potential alterations in collagen fibers can give rise to changes in the ECM weaving, homeostasis and functions. The targets of this thesis work are Lysine Hydroxylases (LHs), a three-member family of multifunctional enzymes involved in collagen lysine modifications, namely hydroxylation and glycosylation. The LH enzymes are considered ECM organizers due to their implication in the collagen maturation pathway and, consequently, alterations in LHs activities lead to malfunctions in ECM and connective tissues, that can result in peculiar genetic syndromes (mutations in PLOD genes) or in tumor metastasis (LH overexpression and/or mislocations). In particular, the focus is on LH2 enzyme: since its activity is strictly related to cellular proliferation, this isoform is shown to be a biomarker of poor prognosis in several solid tumors, and so a very promising druggable target. The aim of this research work is to solve the structure of LH2a, the shorter alternative splicing variant of human LH2 isoform, and use it as a template for a future structure-based drug discovery campaign. Thanks to the strong expertise on collagen enzymes in Prof. Forneris’s lab, we set up and optimized a protocol for the expression, production and purification of the LH2a enzyme. Finally, we performed its biophysical/biochemical characterization exploiting an integrative and orthogonal approach.

Caratterizzazione della lisil idrossilasi umana LH2a, un enzima che modifica il collagene.