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Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal proteins keratin K3 and K12, respectively. To investigate the pathomechanism of this disease we generated and phenotypically characterized a novel knock-in humanised mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation.Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by Western blot, RNA-seq and qRT-PCR. Mass spectrometry and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient.The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, while apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared to the wild-type (p

Corneal cross-linking is nowadays the most used strategy for the treatment of keratoconus and recently it has been exploited for an increasing number of different corneal pathologies, from other ectatic disorders to keratitis. The safety of this technique has been widely assessed, but clinical complications still occur. The potential effects of cross-linking treatment upon the limbus are incompletely understood; it is important therefore to investigate the effect of UV exposure upon the limbal niche, particularly as UV is known to be mutagenic to cellular DNA and the limbus is where ocular surface tumors can develop. The risk of early induction of ocular surface cancer is undoubtedly rare and has to date not been published other than in one case after cross-linking. Nevertheless it is important to further assess, understand, and reduce where possible any potential risk. The aim of this review is to summarize all the reported cases of a pathological consequence for the limbal cells, possibly induced by cross-linking UV exposure, the studies done in vitro or ex vivo, the theoretical bases for the risks due to UV exposure, and which aspects of the clinical treatment may produce higher risk, along with what possible mechanisms could be utilized to protect the limbus and the delicate stem cells present within it.

Subgroup-II aminotransferases (AT-II) are a structural subclass of PLP-dependent enzymes, specialized in transforming for compounds containing an amino group not adjacent to a carboxylate. As the name suggests, the vast majority of these enzymes are in fact transaminases, endowed with some structural peculiarities that help explain their specificity for terminal amines. In this thesis work, we analyzed the specificity of one of the few AT-II enzymes that is not a transaminase, namely O-phosphoethanolamine phospho-lyase lyase (PEA-PL), in terms of both reaction and substrate. This enzyme is quite peculiar for several reasons. First, it is the only known AT-II enzyme (together with its closely related paralog, O-phospho-5-hydroxy-L-lysine phospho-lyase) whose primary function is to catalyze an elimination reaction. Second, because its substrate is, quite unusually, an amine containing a phosphate group. Third, because the enzyme, despite being phylogenetically very close to pyruvate-dependent AT-II transaminases (in particular, to the promiscuous AGXT2) was reported to lack any transaminase activity. The obtained results show that this human PEA-PL is at least 500-fold more active as a lyase than as an aminotransferase and that the lyase reaction is very selective for O-phosphoethanolamine, strongly discriminating against closely related compounds. Dissecting the factors that contribute to such narrow substrate specificity we found that PEA-PL is in fact able to recognize only amines and not amino acids, while the phosphate was shown to be a key determinant of substrate specificity. The results of this first part of the work allowed us to conclude that both the substrate- and reaction specificity of PEA-PL are remarkably strict for a PLP-dependent enzyme. To rationalize these findings, and to better understand their mechanistic basis, we undertook a wide kinetic study encompassing (i) the relative ability of various anionic compounds in inhibiting the PEA-PL reaction; (ii) the pH dependence of the enzyme’s steady-state catalytic parameters and of the inhibitors effectiveness; (iii) a spectroscopic study of the interaction of the enzyme with substrates and inhibitors and (iv) a pre-steady state analysis of the PEA-PL reaction, to gain more information on the formation of catalytic intermediates and generally on the reaction mechanism of the enzyme. The results of these studies confirm that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specificity towards PEA. Single- and multiple-wavelength stopped-flow studies showed that upon reaction with PEA the main absorption band of PLP (lambdamax=412 nm) rapidly blue-shifts to ~400 nm. Further experiments suggested that the newly formed and rather stable 400-nm species most likely represents a Michaelis (non-covalent) complex of PEA with the enzyme. Accumulation of such an early intermediate during turnover is unusual for PLP-dependent enzymes and appears counterproductive for absolute catalytic performance, but it can contribute to optimize substrate specificity. PEA phospho-lyase may hence represent a case of selectivity-efficiency tradeoff. In turn, the strict specificity of the enzyme seems important to prevent inactivation by other amines (structurally resembling PEA) that occur in the brain. Having established the high substrate specificity of PEA-PL, we also decided to explore its practical usefulness, by designing an enzymatic assay for the selective detection of PEA in liquid samples. This assay would be most useful for the clinical laboratory: as PEA levels are increased in in some biological fluids in the course of metabolic, genetic and psychiatric diseases, a method for the rapid and quantitative detection of this analyte would be of relevance to the diagnosis and monitoring of such diseases. The fluorometric assay we begun to develop achieved the desired sensitivity, as it could satisfactorily quantitate micromolar amounts of PEA in a buffer-water system.