Cellular cholesterol homeostasis is controlled by sterol‐regulated proteolysis of membrane‐bound transcription factors called sterol‐regulatory element binding proteins (SREBPs). CPP32, a cysteine protease, was shown previously to cleave SREBP‐1 and SREBP‐2 in vitro at an aspartic acid between the basic helix‐loop‐helix leucine zipper domain and the first trans‐membrane domain, liberating a transcriptionally active fragment. Here, we show that CPP32 exists in an inactive 32 kDa form in Chinese hamster ovary (CHO) cells. When apoptosis was induced with the protein kinase inhibitor staurosporine, CPP32 was cleaved to subunits of 20 and 10 kDa to form the active protease. Under these conditions membrane‐bound SREBP‐1 and SREBP‐2 were both cleaved, and the transcriptionally active N‐terminal fragments were found in nuclear extracts. Similar results were obtained in human U937 cells induced to undergo apoptosis by anti‐Fas and etoposide. The apoptosis‐induced cleavage of SREBPs was not suppressed by sterols, indicating that apoptosis‐induced cleavage and sterol‐regulated cleavage are mediated by different proteases. CHO cells expressing a mutant SREBP‐2 with an Asp–> Ala mutation at the CPP32 cleavage site showed sterol‐regulated cleavage but no apoptosis‐induced cleavage. These data are consistent with the emerging concept that CPP32 is a central mediator in apoptosis. They also indicate that SREBPs, like poly (ADP) ribose polymerase, are cleaved by CPP32 during programmed cell death.

Sterol-resistant CHO cells (SRD-1 cells) fail to repress sterol synthesis and LDL receptor gene transcription when incubated with 25-hydroxycholesterol. Here we trace the defect to a rearrangement in the gene encoding SREBP-2, a membrane-bound transcription factor that regulates cholesterol homeostasis. SREBP-2 is an l139-amino acid protein that is bound to extranuclear membranes via a carboxy-terminal attachment domain. In sterol-depleted cells a protease liberates the amino-terminal fragment (-480 amino acids). This fragment, which contains the transcriptional activation and bHLH-Zip domains, translocates to the nucleus. 25-Hydroxycholesterol abolishes protease activity and halts transcription. SRD-1 cells produce a soluble, truncated form of SREBP-2 (amino acids 1-460) that lacks the membrane attachment domain and activates transcription directly, bypassing the sterol-regulated proteolytic step. Although SRD-1 cells produce full-length SREBP-2 from the wild-type allele and a related transcription factor, SREBP-1, they fail to cleave both of these precursors, indicating that the truncated form of SREBP-2 down-regulates the protease through a form of end-product feedback inhibition. The current data provide genetic evidence for the previously proposed model in which cholesterol homeostasis is controlled by sterol-regulated proteolysis of a membrane-bound bHLH-Zip transcription factor.

The cholesterol analogue 25-hydroxycholesterol kills animal cells by blocking the proteolytic activation of two sterol-regulated transcription factors designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). These proteins, each approximately 1150 amino acids in length, are embedded in the membranes of the nucleus and endoplasmic reticulum by virtue of hydrophobic COOH-terminal segments. In cholesterol-depleted cells the proteins are cleaved to release soluble NH2-terminal fragments of approximately 480 amino acids that enter the nucleus and activate genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. 25-Hydroxycholesterol blocks this cleavage, and cells die of cholesterol deprivation. We previously described a mutant 25-hydroxycholesterol-resistant hamster cell line (SRD-1 cells) in which the SREBP-2 gene had undergone a recombination between the intron following codon 460 and an intron in an unrelated gene. The SREBP-2 sequence terminated at residue 460, eliminating the membrane attachment domain and producing a constitutively active factor that no longer required proteolysis and thus was not inhibited by 25-hydroxycholesterol. Here, we report that two additional sterol-resistant cell lines (SRD-2 and SRD-3) have also undergone genomic rearrangements in the intron following codon 460 of the SREBP-2 gene. Although the molecular rearrangements differ in the three mutant lines, each leads to the production of a constitutively active transcription factor whose SREBP-2 sequence terminates at residue 460. These findings provide a dramatic illustration of the advantage that introns provide in allowing proteins to gain new functions in response to new environmental challenges.

BackgroundGlucagon is an important hormone in the regulation of glucose homeostasis, particularly in the maintenance of euglycemia and prevention of hypoglycemia. In type 2 Diabetes Mellitus (T2DM), glucagon levels are elevated in both the fasted and postprandial states, which contributes to inappropriate hyperglycemia through excessive hepatic glucose production. Efforts to discover and evaluate glucagon receptor antagonists for the treatment of T2DM have been ongoing for approximately two decades, with the challenge being to identify an agent with appropriate pharmaceutical properties and efficacy relative to potential side effects. We sought to determine the hepatic & systemic consequence of full glucagon receptor antagonism through the study of the glucagon receptor knock-out mouse (Gcgr-/-) compared to wild-type littermates.ResultsLiver transcriptomics was performed using Affymetric expression array profiling, and liver proteomics was performed by iTRAQ global protein analysis. To complement the transcriptomic and proteomic analyses, we also conducted metabolite profiling (~200 analytes) using mass spectrometry in plasma. Overall, there was excellent concordance (R = 0.88) for changes associated with receptor knock-out between the transcript and protein analysis. Pathway analysis tools were used to map the metabolic processes in liver altered by glucagon receptor ablation, the most notable being significant down-regulation of gluconeogenesis, amino acid catabolism, and fatty acid oxidation processes, with significant up-regulation of glycolysis, fatty acid synthesis, and cholesterol biosynthetic processes. These changes at the level of the liver were manifested through an altered plasma metabolite profile in the receptor knock-out mice, e.g. decreased glucose and glucose-derived metabolites, and increased amino acids, cholesterol, and bile acid levels.ConclusionsIn sum, the results of this study suggest that the complete ablation of hepatic glucagon receptor function results in major metabolic alterations in the liver, which, while promoting improved glycemic control, may be associated with adverse lipid changes.

PF-04840082 is a humanized prototype anti-Dickkopf-1 (Dkk-1) immunoglobulin isotype G 2 (IgG 2 ) antibody for the treatment of osteoporosis. In vitro, PF-04840082 binds to human, monkey, rat, and mouse Dkk-1 with high affinity. After administration of PF-04840082 to rat and monkey, free Dkk-1 concentrations decreased rapidly and returned to baseline in a dose-dependent manner. In rat and monkey, PF-04840082 exhibited nonlinear pharmacokinetics (PK) and a target-mediated drug disposition (TMDD) model was used to characterize PF-04840082 versus Dkk-1 concentration response relationship. PK/pharmacodynamic (PK/PD) modeling enabled estimation of antibody non-target-mediated elimination, Dkk-1 turnover, complex formation, and complex elimination. The TMDD model was translated to human to predict efficacious dose and minimum anticipated biological effect level (MABEL) by incorporating information on typical IgG 2 human PK, antibody-target association/dissociation rates, Dkk-1 expression, and turnover rates. The PK/PD approach to MABEL was compared with the standard "no adverse effect level" (NOAEL) approach to calculating clinical starting doses and a pharmacological equilibrium method. The NOAEL method gave estimates of dose that were too high to ensure safety of clinical trials. The pharmacological equilibrium approach calculated receptor occupancy (RO) based on equilibrium dissociation constant alone and did not take into account rate of turnover of the target or antibodytarget complex kinetics and, as a result, it likely produced a substantial overprediction of RO at a given dose. It was concluded that the calculation of MABEL according to the TMDD model was the most appropriate means for ensuring safety and efficacy in clinical studies.Osteoporosis is a bone disease characterized by low bone mineral density that leads to bone fragility and, subsequently, bone fractures. The majority of pharmacological osteoporosis therapies, including bisphosphonates, calcitonin, hormone replacement therapy, and selective estrogen receptor modulators, prevent bone loss by reducing bone resorption. Restoration of bone mass in patients suffering from osteoporosis is an area of unmet medical need.Recently, it has been shown that Wnt/low-density lipoprotein receptor-related protein 5 (LRP5) regulates bone mass and activation of Wnt signaling leads to an accrual of bone mass (Gong et al., 2001;Boyden et al., 2002;Little et al., 2002). Wnts are secreted glycoproteins that bind to and activate a receptor complex, which includes LRP5/6 and frizzled proteins. Wnt signaling is tightly regulated by antagonists that include secreted molecules such as Dickkopf-1 (Dkk-1). Binding of Dkk-1 to the Article, publication date, and citation information can be found at