The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.     

Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the -4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.     

Prevalence and intensity of infections of Ascaris lumbricoides and Trichuris trichiura and associated socio-demographic variables in four rural Honduran communities

Between January and March 1998, a cross-sectional survey was carried out in four rural communities in Honduras, Central America. We examined the prevalence and intensity of Ascaris lumbricoides and Trichuris trichiura infections among 240 fecal specimens, and the association between selected socio-demographic variables and infection for 62 households. The overall prevalence of A. lumbricoides and T. trichiura was 45% (95% CI 39.0-51.9) and 38% (95% CI 31.8-44.4) respectively. The most intense infections for Ascaris and Trichuris were found in children aged 2-12 years old. By univariate analysis variables associated with infections of A. lumbricoides were: number of children 2-5 years old (p=0.001), level of formal education of respondents (p=0.01), reported site of defecation of children in households (p=0.02), households with children who had a recent history of diarrhea (p=0.002), and the location of households (p=0.03). Variables associated with both A. lumbricoides and T. trichiura infection included: number of children 6-14 years old (p=0.01, p=0.04, respectively), ownership of a latrine (p=0.04, p=0.03, respectively) and coinfection with either helminth (p=0.001, p=0.001, respectively). By multivariate analysis the number of children 2-5 years living in the household, (p=0.01, odds ratio (OR)=22.2), children with a recent history of diarrhea (p=0.0, OR=39.8), and infection of household members with T. trichiura (p=0.02, OR=16.0) were associated with A. lumbricoides infection. The number of children 6-14 years old in the household was associated with both A. lumbricoides and T. trichiura infection (p=0.04, p=0.01, OR=19.2, OR=5.2, respectively).     

Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.     

Identification of a mutation causing mucopolysaccharidosis type IIIA in New Zealand Huntaway dogs

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disease that occurs due to a deficiency of heparan sulfate sulfamidase (SGSH). The deficiency of SGSH results in the lysosomal accumulation and urinary excretion of the glycosaminoglycan heparan sulfate. The clinical severity of MPS IIIA is predominantly characterized by severe central nervous system degeneration. Naturally occurring MPS IIIA has recently been described in New Zealand Huntaway dogs, with similar disease progression and biochemical characteristics observed in severely affected MPS IIIA patients. Here, we identify the disease-causing mutation in the MPS IIIA Huntaway dog as 708-709insC. The frequency of the 708-709insC mutation in a sample group of 203 New Zealand Huntaway dogs was determined to be 3.8%. The identification of the 708-709insC mutation will permit the identification of heterozygous carriers as an initial step toward establishing a breeding colony of MPS IIIA dogs for the study of various therapeutic strategies targeted to the central nervous system.     

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.     

Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.     

HIV protease as a target for retrovirus vector-mediated gene therapy

The dimeric aspartyl protease of HIV has been the subject of intense research for almost a decade. Knowledge of the substrate specificity and catalytic mechanism of this enzyme initially guided the development of several potent peptidomimetic small molecule inhibitors. More recently, the solution of the HIV protease structure led to the structure-based design of improved peptidomimetic and non-peptidomimetic antiviral compounds. Despite the qualified success of these inhibitors, the high mutation rate associated with RNA viruses continues to hamper the long-term clinical efficacy of HIV protease inhibitors. The dimeric nature of the viral protease has been conducive to the investigation of dominant-negative inhibitors of the enzyme. Some of these inhibitors are defective protease monomers that interact with functional monomers to form inactive protease heterodimers. An advantage of macromolecular inhibitors as compared to small-molecule inhibitors is the increased surface area of interaction between the inhibitor and the target gene product. Point mutations that preserve enzyme activity but confer resistance to small-molecule inhibitors are less likely to have an adverse effect on macromolecular interactions. The use of efficient retrovirus vectors has facilitated the delivery of these macromolecular inhibitors to primary human lymphocytes. The vector-transduced cells were less susceptible to HIV infection in vitro, and showed similar levels of protection compared to other macromolecular inhibitors of HIV replication, such as RevM10. These preliminary results encourage the further development of dominant-negative HIV protease inhibitors as a gene therapy-based antiviral strategy.