Substrate Specificity of Cysteine Proteases Beyond the S Pocket: Mutagenesis and Molecular Dynamics Investigation of Cathepsins L.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity.

Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica.

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F.

A novel mutation in the OAR domain of the ARX gene.

Mutations in ARX gene should be considered in patients with mental disability or/and epilepsy. It is an X-linked gene that has pleiotropic effects. Here, we report the case of a boy diagnosed with Ohtahara syndrome.

[Advances in the identification of the aetiology of mental retardation].

Despite the advances made in the field of genetics, neuroimaging and metabolic diseases, half the children with mental retardation remain without an aetiological diagnosis. A genetic base is estimated to be present in 40% of cases, environmental teratogens and prematurity in 20%, metabolic diseases in 1-5% and multifactor causes in 3-12%. The family history, the detailed medical records required by dysmorphology and the neurological examination will make it possible to establish or suspect a diagnosis in two thirds of the cases and, in the others, scanning tests will be able to confirm an aetiology.

Dissecting the active site of the collagenolytic cathepsin L3 protease of the invasive stage of Fasciola hepatica.

Background: A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2.

Methodology/principal Findings: Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites.

The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep.

Fasciola hepatica M17 leucine aminopeptidase (FhLAP) is thought to play a role in catabolizing peptides generated by the concerted activity of gut-associated endopeptidases on host polypeptides, thus releasing amino acids to be used in protein anabolism. In this study, a recombinant functional form of this homo hexameric metallopeptidase produced in Escherichia coli was used in combination with adjuvants of different types in a vaccination trial in Corriedale sheep against experimental challenge with F. hepatica metacercariae.

Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.

Background: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2.

Methodology/principal Findings: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates.

Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica.

Background: The common liver fluke Fasciola hepatica is the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism.

Characterization of a putative hsp70 pseudogene transcribed in protoscoleces and adult worms of Echinococcus granulosus.

Searching for hsp70 genes in Echinococcus granulosus, a divergent cytoplasmic hsp70-like sequence (EgpsiHsp70) was isolated, possessing a small truncation in the region coding for the C-terminal glycine-rich linker and EEVD-Ct motif. Southern Blot analyses of E. granulosus, and in silico analyses of E.

The major cathepsin L secreted by the invasive juvenile Fasciola hepatica prefers proline in the S2 subsite and can cleave collagen.

Secreted cysteine proteases are major players in host-parasite interactions; in Fasciola hepatica, a distinct group of cathepsins L was found to be predominantly expressed in the juvenile stages, but their enzymatic properties were unknown. Cathepsin L3 (FhCL3) is a main component of the juvenile secretory products and may participate in invasion. To characterize the biochemical properties, the proenzyme was expressed in the methylotrophic yeast Hansenula polymorpha and the mature enzyme was obtained from the culture medium.

A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica.

Secreted cysteine proteases are relevant actors in parasite biology, taking part in critical host colonization roles such as traversing tissue barriers, immune evasion and nutrient digestion. In the trematode Fasciola hepatica, the initial step to successful infection of the mammalian host is the excystment of metacercariae and the invasion through the intestinal wall by the newly excysted juveniles (NEJ). While the cathepsin L-like cysteine proteinases secreted by the adult fluke have been extensively characterized, the cataloguing and description of the cathepsins B and L reported in the invasive stages is only sketchy.

Role of the prosegment of Fasciola hepatica cathepsin L1 in folding of the catalytic domain.

The N-terminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F.