Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway.

Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.

Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor.

Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic ß-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic ß-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in vivo and impaired glucose-stimulated insulin release ex vivo Glucose intolerance was also observed in eight patients with nephrin mutations compared with three patients with other genetic forms of nephrotic syndrome or nine healthy controls.In vitro experiments were conducted to investigate if nephrin affects autocrine signaling through insulin receptor A (IRA) and B (IRB), which are both expressed in human podocytes and pancreatic islets. Coimmunoprecipitation of nephrin and IRB but not IRA was observed and required IR phosphorylation. Nephrin per se was sufficient to induce phosphorylation of p70S6K in an phosphatidylinositol 3-kinase-dependent but IR/Src-independent manner, which was not augmented by exogenous insulin. These results suggest a role for nephrin as an independent modulator of podocyte and pancreatic ß-cell nutrient sensing in the fasting state and the potential of nephrin as a drug target in diabetes.

Sphingomyelinase-like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease.

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVß3 integrin-dependent migration in vitro. Furthermore, αVß3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVß3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVß3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.

Dynamin-mediated Nephrin phosphorylation regulates glucose-stimulated insulin release in pancreatic beta cells.

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function.

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues.

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.

Palliative Care Needs in an Acute Internal Medicine Ward in Mexico.

BACKGROUND: Palliative care is an evolving but underdeveloped practice in Mexico. OBJECTIVE: The primary end point of this prospective observational study was to identify internal medicine inpatients fulfilling advanced criteria within a second-level hospital. Secondary end points were symptom burden, treatment, resource utilization, and one-year survival. DESIGN AND MEASUREMENTS: The 390-sample size calculation was based on previous studies where 15% of inpatients fulfilled palliative care needs. Consecutive admissions were assessed to identify patients with any of the following: cancer, cardiac, renal, hepatic insufficiency, COPD, AIDS, stroke, or fragility until sample size was completed. After obtaining informed consent, interview to patient, attending physician, and chart review was completed to identify any of the following advanced disease criteria in each patient: (1) Surprise question to attending physician of the possibility of the patient dying in the following year, (2) Palliative Performance Scale (PPS) <50, and (3) Advanced disease specific criteria. Interview also included presence of symptoms, functional capacity, and previous resource utilization. Treatment offered was analyzed only on day of admission. One-year follow-up to assess survival was done through the state death certificates. RESULTS: Out of 390 patients, 131 (34%) had any of the diseases studied. Out of 131 patients, 86 (66%) had at least one of the three inclusion criteria for advanced disease. Out of 86 patients, 70 (81%) advanced disease patients died after one-year follow-up. Comparison between patients with no advanced disease (no criteria) versus advanced disease (at least one criteria) showed a significant difference in mean PPS, nutrition status, survival days, inhospital death, weight loss, dependency on activities of daily living, and previous multiple emergency room visits. Advanced disease patients with no death at one year follow-up had significantly more new admissions to that hospital. CONCLUSIONS: The number of patients requiring palliative care in internal medicine wards may be excessive to the current palliative care structures available.

Podocyte-specific GLUT4-deficient mice have fewer and larger podocytes and are protected from diabetic nephropathy.

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.

Cyclodextrin protects podocytes in diabetic kidney disease.

Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD(+)) when compared with diabetic patients with normoalbuminuria (DKD(-)) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.

AT2receptors recruit c-Src, SHP-1 and FAK upon activation by Ang II in PND15 rat hindbrain.

The functional role of AT(2) receptors is unclear and it activates unconventional signaling pathways, which in general do not involve a classical activation of a G-protein. In the present study, we aimed to investigate the transduction mechanism of AT(2) Ang II receptors in PND15 rat hindbrain membrane preparations, which represents a physiological developmental condition. To determine whether Ang II AT(2) receptors induced association to SHP-1 in rat hindbrain, co-immunoprecipitation assays were performed. Stimulation of Ang II AT(2) receptors induced both a transient tyr-phosphorylation and activation of SHP-1. The possible participation of c-Src in Ang II-mediated SHP-1 activation, we demonstrated by recruitment of c-Src in immunocomplexes obtained with anti AT(2) or anti-SHP-1 antibodies. The association of SHP-1 to c-Src was inhibited by PD123319 and the c-Src inhibitor PP2. Similarly, SHP-1 activity determined in AT(2)-immunocomplexes was inhibited by PD123319 and the c-Src inhibitor PP2. Following stimulation with Ang II, AT(2) receptors recruit c-Src, which was responsible for SHP-1 tyr-phosphorylation and activation. Since AT(2) receptors are involved in neuron migration, we tested the presence of FAK in immunocomplexes. Surprisingly, AT(2)-immunocomplexes contained mainly the 85kDa fragment of FAK. Besides, p125FAK associated to SHP-1. In summary, we demonstrated the presence of an active signal transduction mechanism in PND15 rat hindbrain, a developmental stage critical for cerebellar development. In this model, we showed a complex containing AT(2)/SHP-1/c-Src/p85FAK, suggesting a potential role of Ang II AT(2) receptors in cerebellar development and neuronal differentiation.

Role of IRS-4 in PI3-K activation by insulin in HepG2 cells, modulation by Angiotensin II.

Insulin receptor substrate-4 (IRS-4) has a limited tissue expression and its modulation by tyr-phosphorylation is still controversial. We evaluated the participation of IRS-4 in the cross-talk between Angiotensin II (Ang II) and Insulin (Ins) receptors in HepG2 cells. Ins (10(-7)M) induced tyr-phosphorylation of IRS-4 (maximal at 5 min), an effect potentiated by Ang II AT(1) receptors. Phosphatydilinositol-3 kinase (PI3-K) inhibitors Wortmanin or LY294002 reduced Ang II effect on tyr-phosphorylation of IRS-4 to a level comparable to that of Ins alone. Physical association between IRS-4 substrate and PI3-K was demonstrated by co-immunoprecipitation. Recruitment of PI3-K by IRS-4 was induced by Ins (10(-7)M, 5 min) not by Ang II (10(-7)M) and this was inhibited by Wortmanin and LY294002. Ang II did not modify either the association or activation of PI3-K in immunocomplexes. The present data provide novel evidence of IRS-4 phosphorylation mediated by Ins, an effect modulated by Ang II. We report also Ins-induced PI3-K activation mediated by IRS-4. Our findings suggest a role for IRS-4 as a docking protein in the Ins signaling pathway that involves PI3-K association and activation. The present data suggest a possible participation of IRS-4 in cell proliferation Ins-induced.

Angiotensin II modulates tyr-phosphorylation of IRS-4, an insulin receptor substrate, in rat liver membranes.

Angiotensin II (Ang II), a major regulator of blood pressure, is also involved in the control of cellular proliferation and hypertrophy and might exhibit additional actions in vivo by modulating the signaling of other hormones. As hypertension and Insulin (Ins) resistance often coexist and are risk factors for cardiovascular diseases, Ang II and Insulin signaling cross-talk may have an important role in hypertension development. The effect of Ins on protein tyrosine phosphorylation was assayed in rat liver membrane preparations, a rich source of Ins receptors. Following stimulation, Ins (10(-7) M) induced tyr-phosphorylation of different proteins. Insulin consistently induced tyr-phosphorylation of a 160 kDa protein (pp160) with maximum effect between 1 and 3 min. The pp160 protein was identified by anti-IRS-4 but not by anti-IRS-1 antibody. Pre-stimulation with Ang II (10(-7) M) diminishes tyr-phosphorylation level of pp160/IRS-4 in a dose-dependent manner. Okadaic acid, the PP1A and PP2A Ser/Thr phosphatase inhibitor, increases pp160 phosphorylation induced by Ins and prevents the inhibitory effect of Ang II pre-stimulation. Genistein, a tyrosine kinase inhibitor, diminishes tyr-phosphorylation level of IRS-4. PI3K inhibitors Wortmanin and LY294002, both increase tyr-phosphorylation of IRS-4, either in the presence of Ins alone or combined with Ang II. These results suggest that Ins and Ang II modulate IRS-4 tyr-phosphorylation in a PI3K-dependent manner. In summary, we showed that Ins induces tyr-phosphorylation of IRS-4, an effect modulated by Ang II. Assays performed in the presence of different inhibitors points toward a PI3K involvement in this signaling pathway.