The proprotein convertase PCSK9 induces the degradation of low density lipoprotein receptor (LDLR) and its closest family members VLDLR and ApoER2

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.     

Inhibition of Chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the E2 surface glycoprotein

Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus that causes in humans an acute infection characterized by polyarthralgia, fever, myalgia, and headache. Since 2005 this virus has been responsible for an epidemic outbreak of unprecedented magnitude. By analogy with other alphaviruses, it is thought that cellular proteases are able to process the viral precursor protein E3E2 to produce the receptor-binding E2 protein that associates as a heterodimer with E1. Destabilization of the heterodimer by exposure to low pH allows viral fusion and infection. We show that among a large panel of proprotein convertases, membranous furin but also PC5B can process E3E2 from African CHIKV strains at the HRQRR(64) / ST site, whereas a CHIKV strain of Asian origin is cleaved at RRQRR(64) / SI by membranous and soluble furin, PC5A, PC5B, and PACE4 but not by PC7 or SKI-1. Using fluorogenic model peptides and recombinant convertases, we observed that the Asian strain E3E2 model peptide is cleaved most efficiently by furin and PC5A. This cleavage was also observed in CHIKV-infected cells and could be blocked by furin inhibitor decanoyl-RVKR-chloromethyl ketone. This inhibitor was compared with chloroquine for its ability to inhibit CHIKV spreading in myoblast cell cultures, a cell-type previously described as a natural target of this virus. Our results demonstrate the role of furin-like proteases in the processing of CHIKV particles and point out new approaches to inhibit this infection.     

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.     

The role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite Leishmania major

For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH(2)-THF). 5,10-CH(2)-THF can be synthesized from glycine by the mitochondrial glycine cleavage complex (GCC). Bioinformatic analysis revealed the four subunits of the GCC in the L. major genome, and the role of the GCC in parasite metabolism and virulence was assessed through studies of the P subunit (glycine decarboxylase (GCVP)). First, a tagged GCVP protein was expressed and localized to the parasite mitochondrion. Second, a gcvP(-) mutant was generated and shown to lack significant GCC activity using an indirect in vivo assay after incorporation of label from [2-(14)C]glycine into DNA. The gcvP(-) mutant grew poorly in the presence of excess glycine or minimal serine; these studies also established that L. major promastigotes require serine for optimal growth. Although gcvP(-) promastigotes and amastigotes showed normal virulence in macrophage infections in vitro, both forms of the parasite showed substantially delayed replication and lesion pathology in infections of both genetically susceptible or resistant mice. These data suggest that, as the physiology of the infection site changes during the course of infection, so do the metabolic constraints on parasite replication. This conclusion has great significance to the interpretation of metabolic requirements for virulence. Last, these studies call attention in trypanosomatid protozoa to the key metabolic intermediate 5,10-CH(2)-THF, situated at the junction of serine, glycine, and thymidylate metabolism. Notably, genome-based predictions suggest the related parasite Trypanosoma brucei is totally dependent on the GCC for 5,10-CH(2)-THF synthesis.     

The interaction between two Arabidopsis polyadenylation factor subunits involves an evolutionarily-conserved motif and has implications for the assembly and function of the polyadenylation complex

The polyadenylation factor subunit "Factor Interacting with Poly(A) polymerase" (Fip1) is an important bridging subunit in the eukaryotic polyadenylation complex. To better understand the functioning of Fip1 in Arabidopsis, a random combinatorial screen for peptides that interact with a conserved plant-specific domain in the protein was conducted. A search of the Arabidopsis proteome using these Fip1-binding peptides as queries resulted in the identification of a number of putative Fip1-interacting proteins. One of these was the polyadenylation factor subunit, CstF77. This purported interaction was confirmed by yeast two-hybrid and in vitro assays. Mutation of the motif identified in the phage display screen eliminated the interaction, corroborating the results of the phage display screen. The domain of CstF77 that interacts with Fip1 lies at its extreme C-terminus and is distinct from the part of CstF77 that binds CstF64. Taken together, these results suggest that Fip1 is situated near CstF64 in the polyadenylation complex.     

Investigation toward multi-epitope vaccine candidates using native chemical ligation

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.     

Therapeutic attenuation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no current therapy preventing cumulative neuronal loss. There is substantial evidence that mitochondrial dysfunction, oxidative stress, and associated caspase activity underlie the neurodegeneration observed. One potential drug therapy is the potent free radical scavenger and antioxidant cystamine, which has demonstrated significant clinical potential in models of neurodegenerative disorders and human neurological disease. This study examined the oral efficacy of cystamine in the MPTP and 6-hydroxydopamine neurotoxin models of PD. The neuroprotective effects of cystamine treatment significantly ameliorated nigral neuronal loss, preserved striatal dopaminergic projections, and improved striatal dopamine and metabolite levels, as compared to MPTP alone. Cystamine normalized striatal 8-hydroxy-2'-deoxyguanosine levels and ATP concentrations, consistent with reduced oxidative stress and improved mitochondrial function. Cystamine also protected against MPTP-induced mitochondrial loss, as identified by mitochondrial heat shock protein 70 and superoxide dismutase 2, with concomitant reductions in cytochrome c and caspase-3 activities. The neuroprotective value of cystamine was confirmed in the 6-hydroxydopamine model. Together these findings show cystamine's therapeutic benefit to reduce neuronal loss through attenuation of oxidative stress and mitochondrial dysfunction, providing the rationale for human clinical trials in PD patients.     

Complex oligomeric structure of a truncated form of DdrA: a protein required for the extreme radiotolerance of Deinococcus

In order to preserve their genome integrity, organisms have developed elaborate tactics for genome protection and repair. The Deinococcus radiodurans bacteria famous for their extraordinary tolerance toward high doses of radiations or long period of desiccation, possess some specific genes with unknown function which are related to their survival in such extreme conditions. Among them, ddrA is an orphan gene specific of Deinococcus genomes. DdrA, the product of this gene was suggested to be a component of the DNA end protection system. Here we provide a three-dimensional reconstruction of the Deinococcus deserti DdrA((1-160)) by electron microscopy. Although not functional in vivo, this truncated protein keeps its DNA binding ability at the wild-type level. DdrA((1-160)) has a complex three-dimensional structure based on a heptameric ring that can self-associate to form a larger molecular weight assembly. We suggest that the complex architecture of DdrA plays a role in the substrate specificity and favors an efficient DNA repair.     

DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]₁₁ microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol κ) inserts dGMP and dCMP within the [T]₁₁ mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol κ and polymerase α-primase within a [T]₁₁ allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.     

Role of IgE receptors in IgE antibody-dependent cytotoxicity and phagocytosis of ovarian tumor cells by human monocytic cells

Antibodies directed against tumor-associated antigens are emerging as effective treatments for a number of cancers, although the mechanism(s) of action for some are unclear and still under investigation. We have previously examined a chimeric IgE antibody (MOv18 IgE), against the ovarian tumor-specific antigen, folate binding protein (FBP), and showed that it can direct human PBMC to kill ovarian cancer cells. We have developed a three-color flow cytometric assay to investigate the mechanism by which IgE receptors on U937 monocytes target and kill ovarian tumor cells. U937 monocytes express three IgE receptors, the high-affinity receptor, FcεRI, the low-affinity receptor, CD23, and galectin-3, and mediate tumor cell killing in vitro by two mechanisms, cytotoxicity, and phagocytosis. Our results suggest that CD23 mediates phagocytosis, which is enhanced by upregulation of CD23 on U937 cells with IL-4, whereas FcεRI mediates cytotoxicity. We show that effector : tumor cell bridging is associated with both activities. Galectin-3 does not appear to be involved in tumor cell killing. U937 cells and IgE exerted ovarian tumor cell killing in vivo in our xenograft model in nude mice. Harnessing IgE receptors to target tumor cells suggests the potential of tumor-specific IgE antibodies to activate effector cells in immunotherapy of ovarian cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.