Principles of protein-protein interactions.

This review examines protein complexes in the Brookhaven Protein Databank to gain a better understanding of the principles governing the interactions involved in protein-protein recognition. The factors that influence the formation of protein-protein complexes are explored in four different types of protein-protein complexes--homodimeric proteins, heterodimeric proteins, enzyme-inhibitor complexes, and antibody-protein complexes. The comparison between the complexes highlights differences that reflect their biological roles.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.

Dissection of a quantitative trait locus for genetic hypertension on rat chromosome 10.

We have previously identified a locus on rat chromosome 10 as carrying a major hypertension gene, BP/SP-1. The 100:1 odds support interval for this gene extended over a 35-centimorgan (cM) region of the chromosome that included the angiotensin I-converting enzyme (ACE) locus as demonstrated in a cross between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and the normotensive Wistar-Kyoto (WKY-0HD) rat. Here we report on the further characterization of BP/SP-1, using a congenic strain, WKY-1HD. WKY-1HD animals carry a 6-cM chromosomal fragment genotypically identical with SHRSPHD on chromosome 10, 26 cM away from the ACE locus. Higher blood pressures in the WKY-1HD strain compared with the WKY-0HD strain, as well as absence of linkage of the chromosome 10 region to blood pressure in an F2 (WKY-1HD x SHRSPHD) population suggested the existence of a quantitative trait locus, termed BP/SP-1a, that lies within the SHRSP-congenic region in WKY-1HD. Linkage analysis in the F2 (WKY-0HD x SHRSPHD) cross revealed that BP/SP-1a is linked to basal blood pressure, whereas a second locus on chromosome 10, termed BP/SP-1b, that maps closer to the ACE locus cosegregates predominantly with blood pressure after exposure to excess dietary NaCl. Thus, we hypothesize that the previously reported effect of BP/SP-1 represents a composite phenotype that can be dissected into at least two specific components on the basis of linkage data and congenic experimentation. One of the loci identified, BP/SP-1a, represents the most precisely mapped locus affecting blood pressure that has so far been characterized by random-marker genome screening.

Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway.

We report here that the activation of the interleukin 1 beta (IL-1 beta)-converting enzyme (ICE) family is likely to be one of the crucial events of tumor necrosis factor (TNF) cytotoxicity. The cowpox virus CrmA protein, a member of the serpin superfamily, inhibits the enzymatic activity of ICE and ICE-mediated apoptosis. HeLa cells overexpressing crmA are resistant to apoptosis induced by Ice but not by Ich-1, another member of the Ice/ced-3 family of genes. We found that the CrmA-expressing HeLa cells are resistant to TNF-alpha/cycloheximide (CHX)-induced apoptosis. Induction of apoptosis in HeLa cells by TNF-alpha/CHX is associated with secretion of mature IL-1 beta, suggesting that an IL-1 beta-processing enzyme, most likely ICE itself, is activated by TNF-alpha/CHX stimulation. These results suggest that one or more members of the ICE family sensitive to CrmA inhibition are activated and play a critical role in apoptosis induced by TNF.

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.

Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis.

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.

Expressão de metaloproteinases de matriz (MMPS) e de seus inibidores (TIMPS e RECK) em modelo de progressão tumoral de Câncer de mama e sua correlação com dados clínicos-patológicos

O câncer de mama é o tipo de câncer mais comumente detectado em mulheres de todo o mundo. Na maioria das pacientes, a causa de morte se deve, principalmente, à doença metastática que pode se desenvolver a partir do tumor primário. O processo metastático envolve uma complexa cascata de eventos, incluindo a quebra organizada dos componentes da matriz extracelular por metaloproteinases de matriz (MMPs). A atividade das MMPs é precisamente regulada por inibidores específicos, os inibidores teciduais das MMPs (TIMPs). Dado seu papel na progressão tumoral, níveis elevados de MMPs têm sido associados com prognóstico desfavorável para pacientes com câncer. Por outro lado, sendo os TIMPs proteínas multifuncionais, níveis elevados de TlMP-1 e de TIMP-2 correlacionam com agressividade do tumor e prognóstico ruim em diferentes tipos de câncer, incluindo o câncer de mama. O gene supressor de metástase RECK codifica uma glicoproteína de membrana capaz de inibir a invasão e a metástase tumoral através da regulação negativa da atividade de MMPs envolvidas em carcinogênese: MMP-2, MMP-9 e MMP-14 (MT1-MMP). A fim de analisar o papel das MMPs e de seus inibidores (TIMPs e RECK) na progressão tumoral do câncer de mama, o perfil de expressão destes genes foi detectado, através de ensaios de Real-Time PCR, em um painel de cinco linhagens celulares de carcinoma de mama humano com diferentes potenciais invasivos e metastáticos e em 72 amostras teciduais de tumores primários de mama e 30 amostras teciduais de borda normal adjacente ao tumor. O perfil de expressão protéica de RECK foi avaliado em 236 amostras de tumores primários de mama através de ensaios de Tissue Microarray. Além disso, a atividade proteolítica das MMPs foi detectada em ensaios de Zimografia. Os resultados obtidos indicam que a progressão do câncer de mama humano está relacionada com um aumento dos níveis de expressão das MMPs e de seus inibidores específicos. O aumento dos níveis de expressão dos TIMPs parece estar relacionado ao seu papel como proteína multifuncional que pode estar funcionando de maneira a promover, mais do que suprimir, a progressão tumoral. Níveis elevados da expressão protéica de RECK estão associados com pior prognóstico. No entanto, para pacientes em estádios clínicos avançados, altos níveis de expressão de RECK podem estar correlacionados com melhor prognóstico, dependendo do balanço MMP/inibidor. Os níveis de expressão das MMPs apresentaram correlação positiva em relação aos níveis de expressão de seus inibidores específicos, sugerindo a existência de fatores e vias de sinalização comuns envolvidas na regulação coordenada destes genes. Além disso, a síntese do inibidor pode estar relacionada a uma resposta celular ao aumento da expressão e atividade de proteases. O balanço transcricional enzima/inibidor favorece a enzima nas amostras tumorais e, de modo contrário, o inibidor específico nas amostras de borda normal, sugerindo o balanço como o principal fator na determinação da degradação da MEC em processos invasivos e metastáticos. Os resultados obtidos podem contribuir para um melhor entendimento da complexidade dos mecanismos envolvidos na metástase do câncer de mama.

Hypertension and diabetes treatments and risk of adverse outcomes among breast cancer patients

Thesis (Ph.D.)--University of Washington, 2016-06

Secondary prevention of renal and cardiovascular disease: Results of a renal and cardiovascular treatment program in an Australian aboriginal community

Australian Aborigines are experiencing an epidemic of renal and cardiovascular disease. In late 1995 we introduced a treatment program into the Tiwi community, which has a three- to fivefold increase in death rates and a recent annual incidence of treated ESRD of 2760 per million. Eligible for treatment were people with hypertension, diabetics with micro or overt albuminuria, and all people with overt albuminuria. Treatment centered around use of perindopril (Coversyl, Servier), with other agents added to reach BP goals; attempts to control glucose and lipid levels; and health education. Thirty percent of the adult population, or 267 people, were enrolled, with a mean follow up of 3.39 yr. Clinical parameters were followed every 6 mo, and rates of terminal endpoints were compared with those of 327 historical controls matched for baseline disease severity, followed in the pretreatment program era. There was a dramatic reduction in BP in the treatment group, which was sustained through 3 yr of treatment. Albuminuria and GFR stabilized or improved. Rates of natural deaths were reduced by an estimated 50% (P = 0.012); renal deaths were reduced by 57% (P = 0.038); and nonrenal deaths by 46% (P = 0.085). Survival benefit was suggested at all levels of overt albuminuria, and regardless of diabetes status, baseline BP, or prior administration of angiotensin converting enzyme inhibitors (ACEI). No significant benefit was apparent among people without overt albuminuria, nor among those with GFR less than 60 ml/min. An estimated 13 renal deaths and 10 nonrenal deaths were prevented, with the number-needed-to-treat to avoid one terminal event of only 11.6. Falling deaths and renal failure in the whole community support these estimates. The program was extremely cost-effective. Programs like this should be introduced to all high-risk communities as a matter of urgency.

The renin-angiotensin system and male reproduction: new functions for old hormones

The blood-borne renin-angiotensin system (RAS) is known best for its role in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, numerous tissues show intrinsic angiotensin-generating systems that cater for specific local needs through actions that add to, or differ from, the circulating RAS. The male reproductive system has several sites of intrinsic RAS activity. Recent focus on the epididymis, by our laboratories and by others, has contributed important details about the local RAS in this tissue. The RAS components have been localized morphologically and topographically; they have been shown to be responsive to androgens and to hypoxia; and angiotensin has been shown to influence tubular, and consequently, fluid secretion. Components of the RAS have also been found in the testis, vas deferens, prostate and semen. Angiotensin II receptors, type 1 and, to a lesser extent, type 2 are widespread, and angiotensin IV receptors have been localized in the prostate. The roles of the RAS in local processes at these sites are still uncertain and have yet to be fully elucidated, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis and fertilization. Notwithstanding this evidence for the involvement of the RAS in various important aspects of male reproduction, there has so far been a lack of clinical evidence, demonstrable by changes in fertility, for a crucial role of the RAS in male reproduction. However, it is clear that there are several potential targets for manipulating the activity of the male reproductive system by interfering with the locally generated angiotensin systems.

Emerging roles of urotensin-II in cardiovascular disease

Urotensin-II (UII) is a highly potent endogenous peptide within the cardiovascular system. Through stimulation of Galphaq-coupled UT receptors, UII mediates contraction of vascular smooth muscle and endothelial-dependent vasorelaxation, and positive inotropy in human right atrium and ventricle. A pathogenic role of the UT receptor system is emerging in cardiovascular disease states, with evidence for upregulation of the UT receptor system in patients with congestive heart failure (CHF), pulmonary hypertension, cirrhosis and portal hypertension, and chronic renal failure. In vitro and in vivo studies show that under pathophysiological conditions, UII might contribute to cardiomyocyte hypertrophy, extracellular matrix production, enhanced vasoconstriction, vascular smooth muscle cell hyperplasia, and endothelial cell hyper-permeability. Single nucleotide polymorphisms of the UII gene may also impart a genetic predisposition of patients to diabetes. Therefore, the UT receptor system is a potential therapeutic target in the treatment of cardiac, pulmonary, and renal diseases. UT receptor antagonists are currently being developed to prevent and/or reverse the effects of over-activated UT receptors by the endogenous ligand. This review describes UII peptide and converting enzymes, and UT receptors in the cardiovascular system, focusing on pathophysiological roles of UII in the heart and blood vessels. (C) 2004 Elsevier Inc. All rights reserved,

Achieving better in-hospital and after-hospital care of patients with acute cardiac disease

In patients hospitalised with acute coronary syndromes (ACS) and congestive heart failure (CHF), evidence suggests opportunities for improving in-hospital and after hospital care, patient self-care, and hospital-community integration. A multidisciplinary quality improvement program was designed and instigated in Brisbane in October 2000 involving 250 clinicians at three teaching hospitals, 1080 general practitioners (GPs) from five Divisions of General Practice, 1594 patients with ACS and 904 patients with CHF. Quality improvement interventions were implemented over 17 months after a 6-month baseline period and included: clinical decision support (clinical practice guidelines, reminders, checklists, clinical pathways); educational interventions (seminars, academic detailing); regular performance feedback; patient self-management strategies; and hospital-community integration (discharge referral summaries; community pharmacist liaison; patient prompts to attend GPs). Using a before-after study design to assess program impact, significantly more program patients compared with historical controls received: ACS: Angiotensin-converting enzyme (ACE) inhibitors and lipid-lowering agents at discharge, aspirin and beta-blockers at 3 months after discharge, inpatient cardiac counselling, and referral to outpatient cardiac rehabilitation. CHF. Assessment for reversible precipitants, use of prophylaxis for deep-venous thrombosis, beta-blockers at discharge, ACE inhibitors at 6 months after discharge, imaging of left ventricular function, and optimal management of blood pressure levels. Risk-adjusted mortality rates at 6 and 12 months decreased, respectively, from 9.8% to 7.4% (P=0.06) and from 13.4% to 10.1% (P= 0.06) for patients with ACS and from 22.8% to 15.2% (P < 0.001) and from 32.8% to 22.4% (P= 0.005) for patients with CHF. Quality improvement programs that feature multifaceted interventions across the continuum of care can change clinical culture, optimise care and improve clinical outcomes.

Multisite, quality-improvement collaboration to optimise cardiac care in Queensland public hospitals

Objective: To evaluate changes in quality of in-hospital care of patients with either acute coronary syndromes (ACS) or congestive heart failure (CHF) admitted to hospitals participating in a multisite quality improvement collaboration. Design: Before-and-after study of changes in quality indicators measured on representative patient samples between June 2001 and January 2003. Setting: Nine public hospitals in Queensland. Study populations: Consecutive or randomly selected patients admitted to study hospitals during the baseline period (June 2001 to January 2002; n = 807 for ACS, n = 357 for CHF) and post-intervention period (July 2002 to January 2003; n = 717 for ACS, n = 220 for CHF). Intervention: Provision of comparative baseline feedback at a facilitative workshop combined with hospital-specific quality-improvement interventions supported by on-site quality officers and a central program management group. Main outcome measure: Changes in process-of-care indicators between baseline and post-intervention periods. Results: Compared with baseline, more patients with ACS in the post-intervention period received therapeutic heparin regimens (84% v 72%; P < 0.001), angiotensin-converting enzyme inhibitors (64% v 56%; P = 0.02), lipid-lowering agents (72% v 62%; P < 0.001), early use of coronary angiography (52% v 39%; P < 0.001), in-hospital cardiac counselling (65% v 43%; P < 0.001), and referral to cardiac rehabilitation (15% v 5%; P < 0.001). The numbers of patients with CHF receiving β-blockers also increased (52% v 34%; P < 0.001), with fewer patients receiving deleterious agents (13% v 23%; P = 0.04). Same-cause 30-day readmission rate decreased from 7.2% to 2.4% (P = 0.02) in patients with CHF. Conclusion: Quality-improvement interventions conducted as multisite collaborations may improve in-hospital care of acute cardiac conditions within relatively short time frames.

Optimising care of acute coronary syndromes in three Australian hospitals

Objective. To improve quality of in-hospital care of patients with acute coronary syndromes using a multifaceted quality improvement program. Design. Prospective, before and after study of the effects of quality improvement interventions between October 2000 and August 2002. Quality of care of patients admitted between 1 October 2000 and 16 April 2001 (baseline) was compared with that of those admitted between 15 February 2002 and 31 August 2002 (post-intervention). Setting. Three teaching hospitals in Brisbane, Australia. Study participants. Consecutive patients (n = 1594) admitted to hospital with acute coronary syndrome [mean age 68 years (SD 14 years); 65% males]. Interventions. Clinical guidelines, reminder tools, and educational interventions; 6-monthly performance feedback; pharmacist-mediated patient education program; and facilitation of multidisciplinary review of work practices. Main outcome measures. Changes in key quality indicators relating to timing of electrocardiogram (ECG) and thrombolysis in emergency departments, serum lipid measurement, prescription of adjunctive drugs, and secondary prevention. Results. Comparing post-intervention with baseline patients, increases occurred in the proportions of eligible patients: (i) undergoing timely ECG (70% versus 61%; P = 0.04); (ii) prescribed angiotensin-converting enzyme inhibitors (70% versus 60%; P = 0.002) and lipid-lowering agents (77% versus 68%; P = 0.005); (iii) receiving cardiac counselling in hospital (57% versus 48%; P = 0.009); and (iv) referred to cardiac rehabilitation (17% versus 8%; P < 0.001). Conclusions. Multifaceted approaches can improve care processes for patients hospitalized with acute coronary syndromes. Care processes under direct clinician control changed more quickly than those reliant on complex system factors. Identifying and overcoming organizational impediments to quality improvement deserves greater attention.