Infestin 1R, an intestinal subtilisin inhibitor from Triatoma infestans able to impair mammalian cell invasion by Trypanosoma cruzi.

Infestins are Kazal-type serine protease inhibitors described in the midgut of Triatoma infestans, Chagas disease vector. Of all infestins, only infestin 1R (INF1R) does not control host blood coagulation, due to its inhibitory specificity for chymotrypsin-like proteases. We further investigated the effect of INF1R on cell infection by Trypanosoma cruzi. The importance of INF1R reactive site to inhibit T. cruzi cell invasion was confirmed using 1RSFTI, a synthetic cyclic peptide containing the inhibitor reactive site region hybridized to the Sunflower Trypsin Inhibitor-1 (SFTI-1). Our results suggest that INF1R efficiently inhibited parasite cell invasion. For the first time, a serine protease inhibitor, derived from T. infestans, was shown to impair cell invasion by T. cruzi, representing possible new target in parasite cell invasion.

The Relevance of Variants With Unknown Significance for Autism Spectrum Disorder Considering the Genotype-Phenotype Interrelationship.

Several efforts in basic and clinical research have been contributing to unveiling the genetics behind autism spectrum disorders (ASD). However, despite these advancements, many individuals diagnosed with ASD and related neuropsychiatric conditions have been genetically investigated without elucidative results. The enormous genetic complexity of ASD-related conditions makes it a significant challenge to achieve, with a growing number of genes (close to a thousand) involved, belonging to different molecular pathways and presenting distinct genetic variations. Next-generation sequencing (NGS) is the approach most used in genetic research related to ASD, identifying de novo mutation, which is closely related to more severe clinical phenotypes, especially when they affect constrained and loss-of-function intolerant genes. On the other hand, de novo mutation findings contribute to a small percentage of the ASD population, since most of the cases and genetic variants associated with neuropsychiatric conditions are inherited and phenotypes are results of additive polygenic models, which makes statistical efforts more difficult. As a result, NGS investigation can sound vainly or unsuccessful, and new mutations on genes already related with ASD are classified as variants of unknown significance (VUS), hampering their endorsement to a clinical phenotype. This review is focused on currently available strategies to clarify the impact of VUS and to describe the efforts to identify more pieces of evidence throughout clinical interpretation and genetic curation process.

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response.

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.

The first pacifastin elastase inhibitor characterized from a blood sucking animal.

Pacifastin-like protease inhibitors belong to a recent classified protease inhibitor family and they are the smallest protease inhibitors described in animals. In this work, we purified and characterized, for the first time, two neutrophil elastase inhibitors belonging to the pacifastin family from the blood sucking insect Triatoma infestans eggs. The inhibitors showed the same N-terminal sequences, molecular masses of 4257 and 4024Da by MALDI-TOF mass spectrometry and dissociation constants (Ki) for neutrophil elastase of 0.52 and 0.29nM, respectively. Using a fat body cDNA library, we cloned a pacifastin precursor containing two protease inhibitor domains similar to locust pacifastins. The first pacifastin domain translated to T. infestans purified protein, named TIPI1. Recombinant TIPI1 expressed in Pichia pastoris system showed similar inhibitory activities compared to the native inhibitor. Its precursor, called TiPP1, is mainly expressed in fat body, and it is up-regulated after blood feeding. The immune challenges of 1(a) instar T. infestans nymph with bacteria or dsRNA strongly stimulated TiPP1 expression in fat body, suggesting a possible role of TiPP1 in T. infestans immunity. This work is the first to characterize a blood feeding insect pacifastin inhibitor.

Validation of phage display method for protease inhibitor selection using synthetic hybrid peptides.

A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa)) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1'-P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH2; Ki = 14 µM) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases ≈20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH2; Ki = 259 µM). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.

BmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae.

BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. The inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K(i) 0.4 and 0.3nM) and subtilisin A (K(i) 1.4nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K(i) 50nM) and BmSI-6 (K(i) 2.2nM). The BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi.