Mutations within PCSK9 (proprotein convertase subtilisin/ kexin type 9) are associated with dominant forms of familial hyper-and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an ϳ150-fold higher affinity at an acidic endosomal pH (K D ‫؍‬ 4.19 nM) compared with a neutral pH (K D ‫؍‬ 628 nM). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.PCSK9 (proprotein convertase subtilisin/kexin type 9) encodes the ninth member of the mammalian proprotein convertase family of serine endoproteases. PCSK9 is translated as a 692-amino acid proprotein that includes several domains found in other proprotein convertases, including an N-terminal signal sequence, a prodomain, a catalytic domain, and a cysteine-rich C-terminal domain (1-3). The PCSK9 catalytic domain shares high sequence similarity with the proteinase K family of subtilases and contains a catalytic triad (Asp 186 , His 226 , and Ser 386 ) responsible for autoprocessing (1, 4). PCSK9 processing occurs in the secretory pathway, presumably in the endoplasmic reticulum, and results in proteolytic cleavage occurring after Gln 152 (FAQ2SIP). This cleavage generates a stable PCSK9 heterodimer composed of a 14-kDa prodomain fragment and a mature 57-kDa fragment containing the catalytic and C-terminal domains (4, 5). Following processing, the PCSK9 heterodimer exits the ER and is eventually secreted (1). The prodomain of PCSK9 remains strongly bound to the mature protein after secretion, presumably inhibiting the catalytic activity of PCSK9 (1, 5, 6). To date, there is no conclusive evidence that the processed secreted form of PCSK9 can cleave any substrates in a catalytic serine-dependent manner.The first evidence that PCSK9 plays a significant role in regulating plasma low density lipoprotein (LDL) 3 cholesterol (LDL-C) levels was the identification of several missense mutations in PCS...

Previous analyses of NOD mice have shown that some genes control the development of both insulitis and diabetes, while other loci influence diabetes without reducing insulitis. Evidence for the existence of a gene only influencing diabetes, Idd9 on mouse chromosome 4, is provided here by the development of a novel congenic mouse strain, NOD.B10 Idd9. NOD.B10 Idd9 mice display profound resistance to diabetes even though nearly all develop insulitis. Subcongenic analysis has demonstrated that alleles of at least three B10 genes, Idd9.1, Idd9.2, and Idd9.3 are required to produce Idd9-mediated diabetes resistance. Candidate genes with amino acid differences between the NOD and B10 strains have been localized to the 5.6 cM Idd9.2 interval (Tnfr2, Cd30) and to the 2.0 cM Idd9.3 interval (Cd137).

SummaryThe role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca 2 +-activated potassium channels (Pc,c,) and voltage-gated potassium channels (PK,v) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (Ib2 and interferon 3') are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,C~ and Pg.v. we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for Pg, v. Theses studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca2+-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

SummaryThe development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by multiple genes. At least one diabetogenic gene is linked to the major histocompatibility complex (MHC) of the NOD and is most likely represented by the two genes encoding the c~ and chains of the unique NOD class II molecule. Three other diabetogenic loci have recently been identified in the NOD mouse and are located on chromosomes 1, 3, and 11. In addition to the autoimmune diabetes which is caused by destruction of the insulin-producing B cells in the pancreas, other manifestations of autoimmunity are seen in the NOD mouse. These include mononuclear cell inflammation of the submandibular and lacrimal glands, as well as the presence of circulating autoantibodies. To determine the effect of the non-MHC diabetogenic genes on the development of autoimmunity, we constructed the NOD.B10-H-2 b (NOD.H-2 b) strain, which possesses the non-MHC diabetogenic genes from the NOD mouse, but derives its MHC from the C57BL/10 (B10) strain. The NOD.H-2 ~ strain does not develop insulitis, cyclophosphamide-induced diabetes, or spontaneous diabetes. It does, however, develop extensive lymphocytic infiltrates in the pancreas and the submandibular glands that are primarily composed of Thy 1.2 + T cells and B220 + B cells. In addition, autoantibodies are present in NOD.H-2 ~ mice which recognize the "polar antigen" on the insulin-secreting rat tumor line RINm38. These observations demonstrate that the non-MHC genes in the NOD strain, in the absence of the NOD MHC, significantly contribute to the development of autoimmunity. The contribution of a single dose of the NOD MHC to autoimmunity was assessed with a (NOD x NOD.H-2b)F1 cross. Although only '~3% of F1 females developed spontaneous diabetes, approximately 50% of both female and male F1 mice developed insulitis, and 25% of females and 17% of males became diabetic after treatment with cyclophosphamide. These data demonstrate that the MHC-linked diabetogenic genes of the NOD mouse are dominant with decreasing levels of penetrance for the following phenotypes: insulitis > cyclophosphamide-induced diabetes > spontaneous diabetes.T he nonobese diabetic (NOD) 1 mouse spontaneously develops autoimmune diabetes and is an experimental model of human type I diabetes. We previously determined in out-1 Abbreviation used in this paper: NOD, nonobese diabetic. crosses to the C57BL/10 (B10) strain, that at least one gene linked to the MHC of the NOD and three non-MHC-linked recessive diabetogenic genes present in the NOD mouse were required for the development of diabetes (1). Recently, using an outcross with the B10.NOD-H-2g 7 (B10.H-2g 7) strain (a B10 congenic mouse whose MHC was derived from the NOD

A new quantitative cytometric technique, termed the ArrayScan™, is described and used to measure NF-B nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-␣ (TNF␣). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-B was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4 -5 min slower than that of IB␣ degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNF␣ and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNF␣ stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNF␣ stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent IB␣ degradation and subsequent NF-B translocation could also be discriminated; LeuLeu-Leu aldehyde was only a partial inhibitor with an IC 50 of 2 M, while clastolactacystin ␤-lactone was a complete inhibitor with an IC 50 of 10 M. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNF␣ stimulation in both cell types with an IC 50 of 0.4 M. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNF␣ concentration measured after 17 h of stimulation. These results suggest that the ArrayScan™ technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-B. IL-11 and TNF␣ are two master cytokines that induce an almost identical proinflammatory response, including the production of chemotactic cytokines, adhesion molecules, and enzymes such as cyclooxygenase, nitric-oxide synthetase, and matrix metalloproteinases (1, 2). Many of these effects are a result of the activation by both IL-1 and TNF␣ of the NF-B transcription factor pathway, which is associated with the activation of many cellular defense genes (3, 4). Composed of p65 (RelA) and p50 proteins, NF-B is normally present in the cytoplasm in an inactive state in a complex with members of the IB inhibitor protein family, chiefly the 37-kDa IB␣ form. In this complexed form, a nuclear localization sequence found on NF-B is masked by the IB␣, preventing nuclear translocation of NF-B, DNA binding, and subsequent transcriptional activation (5-12). IL-1 or TNF␣ receptor activation induces within several minutes the specific phosphorylation of Ser 32 and Ser 36 on IB␣, the destruction of the phosphorylated IB␣ protein by proteasomes, and the translocation of NF-B to the nucleus (13-1...

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SummaryThe development of type I diabetes in the nonobese diabetic (NOD) mouse is under the control of multiple genes, one or more of which is linked to the major histocompatibility complex (MHC). The MHC class II region has been implicated in disease development, with expression of an I-E transgene in NOD mice shown to provide protection from insulitis and diabetes. To examine the effect of expressing an I-E + or I-E-non-NOD MHC on the NOD background, three I-E + and three I-E-NOD MHC congenic strains (NOD.H-2/s, NOD.H-2 k, and NOD.H-2 h2, and NOD.H-2 h4, NOD.H-2/7, and NOD.H-2 b, respectively) were developed. Of these strains, both I-E + NOD.H-2 h2 and I-E-NOD.H-2 h4 mice developed insulitis, but not diabetes. The remaining four congenic strains were free of insulitis and diabetes. These results indicate that in the absence of the NOD MHC, diabetes fails to develop. Each NOD MHC congenic strain was crossed with the NOD strain to produce I-E + and I-E-F1 mice; these mice thus expressed one dose of the NOD MHC and one dose of a non-NOD MHC on the NOD background. While a single dose of a non-NOD MHC provided a large degree of disease protection to all of the F1 strains, a proportion of I-E + and I-E-F1 mice aged 5-12 mo developed insulitis and cyclophosphamide-induced diabetes. When I-E + F1 mice were aged 9-17 mo, spontaneous diabetes developed as well. These data are the first to demonstrate that I-E + NOD mice develop diabetes, indicating that expression of I-E in NOD mice is not in itself sufficient to prevent insulitis or diabetes. In fact, I-E-F1 strains were no more protected from diabetes than I-E + F1 strains, suggesting that other non-NOD MHC-linked genes are important in protection from disease. Finally, transfer of NOD bone marrow into irradiated I-E + F1 recipients resulted in high incidences of diabetes, indicating that expression of non-NOD MHC products in the thymus, in the absence of expression in bone marrow-derived ceils, is not sufficient to provide protection from diabetes.T he nonobese diabetic (NOD) 1 mouse spontaneously develops autoimmune diabetes (1-4), and is considered an appropriate model for examining the etiology of human type I diabetes. As in human diabetes, the murine disease is associated with lymphocytic infiltration of pancreatic islets (insulitis) (1, 5), the appearance of autoantibodies directed against 3 cell proteins (6-13), the T cell-mediated destruction of 3 cells (14-17), and the presence of both MHC-linked (18-24) and non-MHC-linked (25-30) disease susceptibility genes.As analyzed in an outcross with the C57BL/10SnJ strain, the development of diabetes in the NOD mouse is under poly-1 Abbreviation used in this paper: NOD, nonobese diabetic. genic control (20). At least three non-MHC-linked genes, located on chromosomes 1, 3, and 11 (25, 26), as well as one or more genes in the MHC (18,(20)(21)(22) contribute to disease progression and onset. The MHC class II region has been implicated in disease susceptibility, with the NOD strain expressing a unique I-A B chain (22) a...

C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates "electrostatic" interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.