Fluorescent Location of Human Colonic Tumour Cells by Means of an Enzyme-Inhibitor Complex

Abstract

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

Ddi1-like protein from Leishmania major is an active aspartyl proteinase

Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. We isolated a full-length cDNA encoding a 49-kDa protein from Leishmania major, which exhibited significant deduced amino acid sequence homology with the annotated Leishmania sp. DNA damage-inducible (Ddi1-like) protein, as well as with the Ddi1 protein from Saccharomyces cerevisiae. In contrast to the previously described Ddi1 protein, the protein from L. major displays three domains: (1) an NH2-terminal ubiquitin like; (2) a COOH terminal ubiquitin-associated; (3) a retroviral aspartyl proteinase, containing the typical D[S/T]G signature. The function of the L. major Ddi1-like recombinant protein was investigated after expression in baculovirus/insect cells and biochemical analysis, revealing preferential substrate selectivity for aspartyl proteinase A₂ family substrates, with optimal activity in acidic conditions. The proteolytic activity was inhibited by aspartyl proteinase inhibitors. Molecular modeling of the retroviral domain of the Ddi1-like Leishmania protein revealed a dimer structure that contained a double Asp-Ser-Gly-Ala amino acid sequence motif, in an almost identical geometry to the exhibited by the homologous retroviral aspartyl protease domain of yeast Ddi1 protein. Our results indicate that the isolated Ddi1-like protein is a functional aspartyl proteinase in L. major, opening possibility to be considered as a potential target for novel antiparasitic drugs.     

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.     

Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.     

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

We set out to gain deeper insight into the potential of antibody light chain variable domains (V_Ls) as immunotherapeutics. To this end, we generated a naïve human V_L phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (V_Hs), we isolated a diversity of V_L domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating V_L repertoires are a rich source of non-aggregating domains. In addition, the V_Ls demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human V_Hs revealed that the V_Ls had similar overall profiles with respect to melting temperature (T_m), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of V_Ls did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T_ms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of V_Ls. Human V_Ls and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered V_Ls may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.     

Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.     

Mutation of the Arabidopsis LON2 peroxisomal protease enhances pexophagy

Peroxisomes are critical organelles housing various, often oxidative, reactions. Pexophagy, the process by which peroxisomes are selectively targeted for destruction via autophagy, is characterized in yeast and mammals but had not been reported in plants. In this article, we describe how the peroxisome-related aberrations of a mutant defective in the LON2 peroxisomal protease are suppressed when autophagy is prevented by mutating any of several key autophagy-related (ATG) genes. Our results reveal that plant peroxisomes can be degraded by selective autophagy and suggest that pexophagy is accelerated when the LON2 protease is disabled.     

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman–Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan–Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.     

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.