α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α1-antitrypsin Portland (α1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged α1-PDX (α1-PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin’s active site, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358-↓ or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

Decreased blood pressure response in mice deficient of the α1b-adrenergic receptor

To investigate the functional role of different α1-adrenergic receptor (α1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the α1b-AR (α1b−/−). Reverse transcription–PCR and ligand binding studies were combined to elucidate the expression of the α1-AR subtypes in various tissues of α1b +/+ and −/− mice. Total α1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the α1b −/− as compared with +/+ mice. Because of the large decrease of α1-AR in the heart and the loss of the α1b-AR mRNA in the aorta of the α1b−/− mice, the in vivo blood pressure and in vitro aorta contractile responses to α1-agonists were investigated in α1b +/+ and −/− mice. Our findings provide strong evidence that the α1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by α1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in α1b −/− as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in α1b−/− mice. The α1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different α1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Relationships of dehydroepiandrosterone sulfate in the elderly with functional, psychological, and mental status, and short-term mortality: A French community-based study

In human beings of both sexes, dehydroepiandrosterone sulfate (DHEAS) circulating in blood is mostly an adrenally secreted steroid whose serum concentration (in the micromolar range and 30–50% higher in men than in women) decreases with age, toward ≈20–10% of its value in young adults during the 8th and 9th decades. The mechanism of action of DHEA and DHEAS is poorly known and may include partial transformation into sex steroids, increase of bioavailable insulin-like growth factor I, and effects on neurotransmitter receptors. Whether there is a cause-to-effect relationship between the decreasing levels of DHEAS with age and physiological and pathological manifestations of aging is still undecided, but this is of obvious theoretical and practical interest in view of the easy restoration by DHEA administration. Here we report on 622 subjects over 65 years of age, studied for the 4 years since DHEAS baseline values had been obtained, in the frame of the PAQUID program, analyzing the functional, psychological, and mental status of a community-based population in the south-west of France. We confirm the continuing decrease of DHEAS serum concentration with age, more in men than in women, even if men retain higher levels. Significantly lower values of baseline DHEAS were recorded in women in cases of functional limitation (Instrumental Activities of Daily Living), confinement, dyspnea, depressive symptomatology, poor subjective perception of health and life satisfaction, and usage of various medications. In men, there was a trend for the same correlations, even though not statistically significant in most categories. No differences in DHEAS levels were found in cases of incident dementia in the following 4 years. In men (but not in women), lower DHEAS was significantly associated with increased short-term mortality at 2 and 4 years after baseline measurement. These results, statistically established by taking into account corrections for age, sex, and health indicators, suggest the need for further careful trials of the administration of replacement doses of DHEA in aging humans. Indeed, the first noted results of such “treatment” are consistent with correlations observed here between functional and psychological status and endogenous steroid serum concentrations.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Hsp90 Is Required for Pheromone Signaling in Yeast

The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature. Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies. We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses. We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90. Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90β instead of the wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding yeast, Ste11 represents the first identified endogenous “substrate” of Hsp90. Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90β are specifically defective for the former and the latter, respectively. These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways.

New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (λs = 5), twin studies, and segregation analysis. HLA, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA (P < 2.5⋅10−5) and nominal for 19 markers in 14 other regions (P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13, and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22–23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0.001 and suggested an interaction between this putative RA locus and HLA. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC50 values of 18–100 nM in a standard TRAP assay.

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

We have determined the solution structure of the C-terminal quarter of human poly(A)-binding protein (hPABP). The protein fragment contains a protein domain, PABC [for poly(A)-binding protein C-terminal domain], which is also found associated with the HECT family of ubiquitin ligases. By using peptides derived from PABP interacting protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a peptide binding domain. We use chemical shift perturbation analysis to identify the peptide binding site in PABC and the major elements involved in peptide recognition. From comparative sequence analysis of PABC-binding peptides, we formulate a preliminary PABC consensus sequence and identify human ataxin-2, the protein responsible for type 2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.

Prospective measurements of dehydroepiandrosterone sulfate in a cohort of elderly subjects: Relationship to gender, subjective health, smoking habits, and 10-year mortality

The decrease with age of the adrenal-secreted dehydroepiandrosterone sulfate (DHEAS) in serum has suggested that it may be causally related to longevity. For the PAQUID [People (Personnes) Aged (Agées) About What (Quid, in Latin)] cohort of elderly subjects, we have previously reported higher DHEAS in men than in women, a decrease with age and, among men, a negative correlation between the DHEAS level and mortality at 2 and 4 years. Here, with an 8-year followup in 290 subjects, we show a global decrease of 2.3% per year for men and 3.9% per year for women. However, in approximately 30% of cases, there was an increase of DHEAS. We observed no relationship between the evolution of DHEAS level and functional, psychological, and mental status, possibly because of selection by death. In women, no association was found between mortality and DHEAS level. In men, the relative risk (RR) of death was higher for the lowest levels of DHEAS (RR = 1.9, P = 0.007), with RR = 6.5, P = 0.003 for those under 70 years old, a result indicating heterogeneity of the population. There was an effect of subjective health on mortality that disappeared after adjustment of DHEAS levels, suggesting its relation with these DHEAS levels. Death RR was much higher in smokers with a low DHEAS level than in nonsmokers with high DHEAS (RR = 6.7, P = 0.001). We submit that the involvement of DHEAS is possibly different according to gender, that association between low DHEAS level and mortality only for men under 70 years old possibly reflects heterogeneity of the population, and that DHEAS level is a reliable predictor of death in male smokers.