Cells infected with prions contain both prion protein isoforms cellular prion protein (PrP C ) and scrapie prion protein (PrP Sc ). PrPSc is formed posttranslationally through the pathological refolding of PrP C . In scrapieinfected ScN2a cells, the metabolism of both PrP isoforms involves cholesterol-dependent pathways. We show here that both PrP C and PrP Sc are attached to Triton X-100-insoluble, low-density complexes or "rafts." These complexes are sensitive to saponin and thus probably contain cholesterol. This finding suggests that the transformation PrP C 3 PrP Sc occurs within rafts. It also reveals the existence of rafts in late compartments of the endocytic pathway, where most PrP Sc resides. When Triton X-100 lysates of cells were incubated at 37°C prior to density analysis, PrP C was still found in buoyant complexes, although it now failed to sediment at high speed. This property was shared by another glycophosphatidyl inositol protein, Thy-1, and also by the raft resident GM1. In one ScN2a clone and in the brain of a Syrian hamster with scrapie, Triton X-100 extraction at 37°C permitted resolution of PrP C and PrP Sc into two distinct peaks of different densities. This suggests that there are two populations of PrP-containing rafts and may permit isolation of PrP C -specific rafts from those containing PrP Sc . Our findings reinforce the contention that rafts are involved in various aspects of PrP metabolism and in the "life cycle" of prions.Prions are unique proteinaceous pathogens that cause a series of fatal encephalopathies such as Creutzfeldt-Jakob disease of humans, scrapie of sheep, and bovine spongiform encephalopathy (1). Prions seem to propagate in the host by posttranslationally (2, 3) refolding a normal host protein, the cellular prion protein (PrP C ), 1 to an aberrant conformation (4, 5). The only known component of prions is the misfolded isoform of PrP C , the scrapie prion protein (PrP Sc ) (6, 7). Current evidence argues that direct interaction of PrP Sc with PrP C is a prerequisite for the transformation PrP C ϩ PrP Sc 3 2PrP Sc (8,9). PrP C is a phosphoinositol glycolipid (GPI)-anchored glycoprotein present on the surface of neurons and other cells (10,11). The PrP isoforms appear to be chemically identical (12) but differ in their conformation (4); PrP C contains ϳ40% ␣-helix and is devoid of ␤-sheet, whereas PrP Sc has more than 40% ␤-sheet (4, 13-16). The two PrP isoforms differ considerably in their properties; PrP C is readily soluble in most detergents and is completely degraded by proteases, whereas PrP Sc is insoluble in detergents, possesses a protease-resistant core termed PrP27-30, and polymerizes into amyloidic structures called prion rods (17,18). Since no isoform-specific PrP antibody has yet been developed, the disparate properties of PrP C and PrP Sc serve as the sole ways to differentiate experimentally between these proteins. The subcellular sites where PrP Sc is formed, and the trafficking pathways leading to these sites, remain largely unknown. Scrapie-infecte...

Mechanisms of progression of chronic renal diseases, a major healthcare burden, are poorly understood. Angiotensin II (AngII), the major renin-angiotensin system effector, is known to be involved in renal deterioration, but the molecular pathways are still unknown. Here, we show that mice overexpressing a dominant negative isoform of epidermal growth factor receptor (EGFR) were protected from renal lesions during chronic AngII infusion. Transforming growth factor-alpha (TGF-alpha) and its sheddase, TACE (also known as ADAM17), were induced by AngII treatment, TACE was redistributed to apical membranes and EGFR was phosphorylated. AngII-induced lesions were substantially reduced in mice lacking TGF-alpha or in mice given a specific TACE inhibitor. Pharmacologic inhibition of AngII prevented TGF-alpha and TACE accumulation as well as renal lesions after nephron reduction. These findings indicate a crucial role for AngII-dependent EGFR transactivation in renal deterioration and identify in TACE inhibitors a new therapeutic strategy for preventing progression of chronic renal diseases.

Mechanisms of progression of chronic kidney disease (CKD), a major health care burden, are poorly understood. EGFR stimulates CKD progression, but the molecular networks that mediate its biological effects remain unknown. We recently showed that the severity of renal lesions after nephron reduction varied substantially among mouse strains and required activation of EGFR. Here, we utilized two mouse strains that react differently to nephron reduction -FVB/N mice, which develop severe renal lesions, and B6D2F1 mice, which are resistant to early deterioration -coupled with genome-wide expression to elucidate the molecular nature of CKD progression. Our results showed that lipocalin 2 (Lcn2, also known as neutrophil gelatinase-associated lipocalin [NGAL]), the most highly upregulated gene in the FVB/N strain, was not simply a marker of renal lesions, but an active player in disease progression. In fact, the severity of renal lesions was dramatically reduced in Lcn2 -/-mice. We discovered that Lcn2 expression increased upon EGFR activation and that Lcn2 mediated its mitogenic effect during renal deterioration. EGFR inhibition prevented Lcn2 upregulation and lesion development in mice expressing a dominant negative EGFR isoform, and hypoxia-inducible factor 1α (Hif-1α) was crucially required for EGFR-induced Lcn2 overexpression. Consistent with this, cell proliferation was dramatically reduced in Lcn2 -/-mice. These data are relevant to human CKD, as we found that LCN2 was increased particularly in patients who rapidly progressed to end-stage renal failure. Together our results uncover what we believe to be a novel function for Lcn2 and a critical pathway leading to progressive renal failure and cystogenesis.

The importance of post-transcriptional regulation by small non-coding RNAs has recently been recognized in both pro-and eukaryotes. Small RNAs (sRNAs) regulate gene expression posttranscriptionally by base pairing with the mRNA. Here we use dynamical simulations to characterize this regulation mode in comparison to transcriptional regulation mediated by protein-DNA interaction and to post-translational regulation achieved by protein-protein interaction. We show quantitatively that regulation by sRNA is advantageous when fast responses to external signals are needed, consistent with experimental data about its involvement in stress responses. Our analysis indicates that the half-life of the sRNA-mRNA complex and the ratio of their production rates determine the steady-state level of the target protein, suggesting that regulation by sRNA may provide fine-tuning of gene expression. We also describe the network of regulation by sRNA in Escherichia coli, and integrate it with the transcription regulation network, uncovering mixed regulatory circuits, such as mixed feed-forward loops. The integration of sRNAs in feedforward loops provides tight repression, guaranteed by the combination of transcriptional and post-transcriptional regulations.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (HRIs) have been recently shown to prevent atherosclerosis progression. Clinical benefit results from combined actions on various components of the atherosclerotic lesion. This study was designed to identify the effects of HRI on one of these components, the endothelial fibrinolytic system. Aortas isolated from rats treated for 2 days with lovastatin (4 mg/kg body wt per day) showed a 3-fold increase in tissue plasminogen activator (tPA) activity. In a rat aortic endothelial cell line (SVARECs) and in human nontransformed endothelial cells (HUVECs), HRI induced an increase in tPA activity and antigen in a time- and concentration-dependent manner. In SVARECs, the maximal response was observed when cells were incubated for 48 hours with 50 micromol/L HRI. An increase of tPA mRNA was also in evidence. In contrast, HRI inhibited plasminogen activator inhibitor-1 activity and mRNA. The effects of HRI were reversed by mevalonate and geranylgeranyl pyrophosphate, but not by LDL cholesterol and farnesyl pyrophosphate, and were not induced by alpha-hydroxyfarnesyl phosphonic acid, an inhibitor of protein farnesyl transferase. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and plasminogen activator inhibitor-1 activity and blocked its reversal by geranylgeranyl pyrophosphate. The effect of HRI was associated with a disruption of cellular actin filaments without modification of microtubules. A disrupter of actin filaments, cytochalasin D, induced the same effect as lovastatin on tPA, whereas a disrupter of microtubules, nocodazole, did not. In conclusion, HRI can modify the fibrinolytic potential of endothelial cells, likely via inhibition of geranylgeranylated Rho protein and disruption of the actin filaments. The resulting increase of fibrinolytic activity of endothelial cells may contribute to the beneficial effects of HRI in the progression of atherosclerosis.

Heterozygous mutations in the NPT2a gene may be responsible for hypophosphatemia and urinary phosphate loss in persons with urolithiasis or bone demineralization.

Our results suggest that the mTORC pathway is involved in the vascular lesions associated with the antiphospholipid syndrome. (Funded by INSERM and others.).

In chronic kidney disease (CKD), loss of functional nephrons results in metabolic and mechanical stress in the remaining ones, resulting in further nephron loss. Here we show that Akt2 activation has an essential role in podocyte protection after nephron reduction. Glomerulosclerosis and albuminuria were substantially worsened in Akt2(-/-) but not in Akt1(-/-) mice as compared to wild-type mice. Specific deletion of Akt2 or its regulator Rictor in podocytes revealed that Akt2 has an intrinsic function in podocytes. Mechanistically, Akt2 triggers a compensatory program that involves mouse double minute 2 homolog (Mdm2), glycogen synthase kinase 3 (Gsk3) and Rac1. The defective activation of this pathway after nephron reduction leads to apoptosis and foot process effacement of the podocytes. We further show that AKT2 activation by mammalian target of rapamycin complex 2 (mTORC2) is also required for podocyte survival in human CKD. More notably, we elucidate the events underlying the adverse renal effect of sirolimus and provide a criterion for the rational use of this drug. Thus, our results disclose a new function of Akt2 and identify a potential therapeutic target for preserving glomerular function in CKD.