Proprotein convertase subtilisin/kexin 9 (PCSK9) is a key regulator of lipid metabolism by degrading liver LDL receptors. Structural studies have provided molecular details of PCSK9 function. However, the N-terminal acidic stretch of the PCSK9 prodomain (Q31–T60) has eluded structural investigation, since it is in a disordered state. The interest in this region is intensified by the presence of human missense mutations associated with low and high LDL-c levels (E32K, D35Y, and R46L, respectively), as well as two posttranslationally modified sites, sulfated Y38 and phosphorylated S47. Herein we show that a segment within this region undergoes disorder-to-order transition. Experiments with acidic stretch-derived peptides demonstrated that the folding is centered at the segment Y38–L45, which adopts an α-helix as determined by NMR analysis of free peptides and by X-ray crystallography of peptides in complex with antibody 6E2 (Ab6E2). In the Fab6E2–peptide complexes, the structured region features a central 2 1/4-turn α-helix and encompasses up to 2/3 of the length of the acidic stretch, including the missense mutations and posttranslationally modified sites. Experiments with helix-breaking proline substitutions in peptides and in PCSK9 protein indicated that Ab6E2 specifically recognizes the helical conformation of the acidic stretch. Therefore, the observed quantitative binding of Ab6E2 to native PCSK9 from various cell lines suggests that the disorder-to-order transition is a true feature of PCSK9 and not limited to peptides. Because the helix provides a constrained spatial orientation of the missense mutations and the posttranslationally modified residues, it is probable that their biological functions take place in the context of an ordered conformational state.

Recent years have witnessed an explosive growth of the World-Wide Web with an increasing number of images corporated in a Web page. Images was mentioned to constitue upto 70% of the Internet traffic and are currently used on the Web for various purposes such as text-related content, decoration, navigation, and advertisement. Unfortunately, not all images contain valuable information for most users. For instance, advertisements, banners, or decorative images are typically not the reason for most users to visit a particular Web site. While the information value of these images appear small, they consume network bandwidth and increase service delays. This paper discusses an application service that enhances the quality of information on a Web page. The service allows a user to specify desirable categories of images to retain in a page. The service automatically detects image purposes associated with the requested Web page and renders the page with only the desirable images.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a prominent role in normal vascular biology and pathology. In an experimental wound model, the mechanical disruption of monolayers of cultured endothelial cells resulted in two phenotypically distinct cell subpopulations in which VEGF was internalized by alternative endocytotic pathways and delivered to different subcellular compartments. In the cells away from the wound, VEGF was internalized via the classical receptor-mediated endocytosis pathway and accumulated in the endosomal compartment, whereas in the cells situated at the edges of a wound, VEGF was rapidly taken up and translocated to the nucleus, VEGF internalization and subsequent nuclear accumulation only occurred for a short period of time after the wounding and was specifically abolished by antibodies that bind to the KDR binding site of VEGF.In the cells with VEGF nuclear accumulation, the levels of wound healing related proteins, such as Factor VIII (FVIII), tissue factor (TF) and tissue plasminogen activator, rapidly and dramatically increased compared to the cells that internalized VEGF via the classical endocytotic pathway. The increase in FVIII and TF was abolished when the nuclear transport is blocked. These data suggest that nuclear VEGF accumulation may be involved in modulating the levels of the proteins of the coagulation and fibrinolysis pathways.

Uncoupling proteins (UCPs) are a family of mitochondrial transporter proteins that have been implicated in thermoregulatory heat production and maintenance of the basal metabolic rate. We have identified and partially characterized a novel member of the human uncoupling protein family, termed uncoupling protein‐4 (UCP4). Protein sequence analyses showed that UCP4 is most related to UCP3 and possesses features characteristic of mitochondrial transporter proteins. Unlike other known UCPs, UCP4 transcripts are exclusively expressed in both fetal and adult brain tissues. UCP4 maps to human chromosome 6p11.2–q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain.

Because of an error in production, the relative contributions of the above authors were printed incorrectly in the original article. They are given correctly in the footnote below.

Although considerable evidence implicates insulin-degrading enzyme (IDE) in the cellular metabolism of insulin in many cell types, its mechanism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme's peroxisomal location and its ability to degrade insulin by mutation of its peroxisomal targeting signal (PTS), the carboxy terminal A/S-K-L tripeptide. Site-directed mutagenesis was used to destroy the peroxisomal targeting signal of human insulin-degrading enzyme by changing alanine to leucine (AL.pts), leucine to valine (LV.pts), or by deleting the entire tripeptide (DEL.pts). The alanine or leucine mutants, when expressed in COS cells, were indistinguishable from wild-type insulin-degrading enzyme with respect to size (110 kDa), amount of immunoreactive material, ability to bind insulin, in vitro activity, and cellular degradation of insulin. In contrast, the deletion mutant was shorter in size (approximately 0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme's carboxy terminus appeared to confer enzyme stability, the conserved sequence was not required for insulin degradation. Finally, an immunocytofluorescence study showed that, whereas a significant amount of the wild-type protein was localized in peroxisomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzyme does not require peroxisomal localization for insulin degradation and suggest that this enzyme has multiple cellular functions.

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

When added to cultured opossum kidney cells, IGF-I is internalized and transported to distinct intracellular compartments that depend on the cell location within the monolayer. In resting cells away from the periphery of the monolayer, IGF-I is internalized by a clathrin coated pit pathway and delivered to the endosomal compartment. In contrast, cells growing at the edges of a monolayer or an experimental wound internalize IGF-I by an alternative route which rapidly delivers IGF-I to the nucleus. Similarly to IGF-I, IGFBP-3 is also internalized and accumulates in the endosomal compartment in resting cells whereas it is targeted to the nucleus in proliferating cells. IGFBP-3, which contains a putative nuclear targeting signal, may act as a carrier for IGF-I nuclear transport. The transport of IGF-I and IGFBP-3 to two different compartments may influence their biological activity.

Heregulins (neuregulins) are a family of proteins known to interact and activate the receptor tyrosine kinases ErbB2 in association with ErbB3 or ErbB4. Using immunofluorescence microscopy, electron microscopy autoradiography, and SDS-PAGE analysis of nuclear fractions, we show that the heregulin-beta1(1-244) isoform is rapidly internalized and translocated to the nucleus of SK-BR-3 breast cancer cells as an intact molecule. Heregulin-beta 1(1-244) treatment up-regulated expression of c-myc mRNA and protein, which was also observed to undergo its own translocation from the cytosol to the nucleus. c-myc thus appears to be a cellular target gene of HRGbeta 1(1-244), and its induction may be related to the nuclear translocation of heregulin.

Human erythrocytes infected with the malarial parasite Plasmodium falciparum contain flattened membrane lamellae, It has been suggested that the lamellae may be involved in the sorting of malarial proteins to the cytoplasm and the cell membrane of the host erythrocyte. We have previously shown that the lamellae accumulate sphingolipids by virtue of their lipid composition in a manner similar to the trans-Golgi and the trans-Golgi network in mammalian cells, In this paper, we show by immunofluorescence microscopy that a monoclonal antibody to the lamellae labeled a perinuclear organelle that colocalized with WGA and the mannose-6-phosphate receptor in cultured mammalian cells, Immunoelectron microscopy experiments revealed that LWLI labels cisternae of the trans-face and the trans-Golgi network, Western blot analysis of subcellular fractions using LWLI detected a 170 kD protein which is associated with the luminal side of Golgi membranes of rat liver and is conserved in all cell lines studied, Our results indicate that (i) the 170 kD protein is a novel marker of the mammalian trans-Golgi and the trans-Golgi network and (ii) in addition to similarities in their morphological and lipid characteristics, the lamellae induced by P. falciparum in erythrocytes share proteinaceous determinants with the Golgi apparatus of mammalian cells.

A monoclonal antibody, LWLI, recognized 3 proteins of 45, 50 and 102 kDa inPlasmodium falciparum-infected erythrocytes. The 45- and 50-kDa proteins were parasite-encoded and displayed markedly different peptide maps, indicating that they were distinct plasmodial polypeptides with a common antigenic epitope rather than differentially processed forms of a primary translational product. The 45-kDa protein was present throughout intraerythrocytic growth, while the 50-kDa molecule was not detected earlier than 11 h in the life cycle. The 102-kDa protein was only expressed in trophozoite- and schizont-infected red cells: its structural relationship to the 45- and 50-kDa proteins, if any, remains undefined. By indirect immunofluorescence and immunoelectron microscopy, LWLI bound to flattened intraerythrocytic cisternae exported into the erythrocyte cytoplasm. The results support the theory that proteins recognized by the antibody were concentrated in these compartments and their common antigenic epitope may serve as a marker for the cisternae. Stage-specific expression of LWLI reactive proteins implicates developmental regulation of cisternal functions during asexual parasite development.

We have examined the accumulation and metabolism ofN-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) inPlasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2°C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2°C, resulted in a depletion of the ceramide label from the red cell membrane and an accumulation of fluorescence in parasite membranes, by an energy independent process. When the cells were subsequently warmed to 37°C for 30 min, virtually all of the ceramide was converted toN-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine-1-phosphocholine (C6-NBD-Sm). Uninfected erythrocytes were incapable of sphingomyelin synthesis. By fluorescence microscopy, sphingomyelin synthesis in infected erythrocytes occurred in compartments morphologically similar to those accumulating ceramide. To examine the intracellular sites of ceramide accumulation glutaraldehyde fixed cells were labeled with C6-NBD-ceramide and subsequently back extracted to remove excess probe. This resulted in a depletion of label at the red cell membrane but prominent fluorescence remained associated with the parasite. Photobleaching in the presence of diaminobenzidine resulted in precipitates in intraerythrocytic cisternae and the vacuolar membrane surrounding the parasite, rather than a perinuclear Golgi apparatus within the organism. The results support a novel organisation of plasmodial membranes regulating the accumulation and metabolism of C6-NBD-cer in infected erythrocytes.