Amphipathic CRAC-Containing Peptides Derived from the Influenza Virus A M1 Protein Modulate Cholesterol-Dependent Activity of Cultured IC-21 Macrophages

Entry of many viral and bacterial pathogens into host cells depends on cholesterol and/or cholesterol-enriched domains (lipid rafts) in the cell membrane. Earlier, we showed that influenza virus A matrix protein M1 contains amphipathic α-helices with exposed cholesterol-recognizing amino acid consensus (CRAC) motifs. In order to test possible functional activity of these motifs, we studied the effects of three synthetic peptides corresponding to the CRAC-containing α-helices of the viral M1 protein on the phagocytic activity of cultured mouse IC-21 macrophages. The following peptides were used: LEVLMEWLKTR (M1 α-helix 3, a.a. 39–49; further referred to as peptide 1), NNMDKAVKLYRKLK (M1 α-helix 6, a.a. 91–105; peptide 2), and GLKNDLLENLQAYQKR (M1 α-helix 13, a.a. 228–243; peptide 3). We found that all three peptides modulated interactions of IC-21 macrophages with non-opsonized 2-μm target particles. The greatest effect was demonstrated by peptide 2: in the presence of 35 μM peptide 2, the phagocytic index of IC-21 macrophages exceeded the control value by 60%; 10–11 mM methyl-β-cyclodextrin abolished this effect. Peptides 1 and 3 exerted weak inhibitory effect in a narrow concentration range of 5–10 μM. The dose-response curves could be approximated by a sum of two (stimulatory and inhibitory) components with different Hill coefficients, suggesting existence of at least two peptide-binding sites with different affinities on the cell surface. CD spectroscopy confirmed that the peptides exhibit structural flexibility in solutions. Altogether, our data indicate that amphipathic CRAC-containing peptides derived from the viral M1 protein modulate lipid raft-dependent processes in IC-21 macrophages.     

Virus-cell fusion inhibitory activity of novel analogue peptides based on the HP (2-20) derived from N-terminus of Helicobacter pylori Ribosomal Protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antibacterial sequence derived from N-terminus of Helicobacter pylori Ribosomal Protein L1 (RPL1). It has a broad-spectrum microbicidal activity in vitro that is thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that HP (2-20) might be possessed virus-cell fusion inhibitory activity. To develop the novel virus-cell fusion inhibitory peptides, several analogues with amino acid substitution were designed to increase or decrease only net hydrophobic region. In particular, substitution of Gln and Asp for hydrophobic amino acid, Trp at position 17 and 19 of HP (2-20) (Anal 3) caused a dramatic increase in virus-cell fusion inhibitory activity without hemolytic effect.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) with Antibodies against Synthetic Peptides Derived from Penicillin-Binding Protein 2′

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin-binding protein 2′ (PBP2′) of methicillin-resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2′ of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin-susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2′ was developed using these antibodies. The method could detect PBP2′ extracted from as few as 3 × 10⁴ cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio-count was observed. IRMA was positive for all 51 methicillin-resistant staphylococci isolated from patients, and was negative for all the 28 methicillin-susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.     

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.     

Studies on Pea Ribosomal Proteins: Conformational and Biological Activity Changes of Ribosomal Subunits Derived by NH(4)Cl Dissociation

Ribosomal subunits prepared by NH(4)Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L(2) and L(9) as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH(4)Cl retained L(2) and L(9), but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl(2)) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH(4)Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L(2) and L(9), showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH(4)Cl (this apparently leads to a preferential detachment of L(2) and L(9) at 0.5 m NH(4)Cl) and that the activity of the purified subunits depends not only on the presence of L(2) and L(9) but also on the organization of these proteins within the 60S subunits.     

Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.     

Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease

In Alzheimer disease (AD) and frontotemporal dementia the microtubule-associated protein Tau becomes progressively hyperphosphorylated, eventually forming aggregates. However, how Tau dysfunction is associated with functional impairment is only partly understood, especially at early stages when Tau is mislocalized but has not yet formed aggregates. Impaired axonal transport has been proposed as a potential pathomechanism, based on cellular Tau models and Tau transgenic mice. We recently reported K369I mutant Tau transgenic K3 mice with axonal transport defects that suggested a cargo-selective impairment of kinesin-driven anterograde transport by Tau. Here, we show that kinesin motor complex formation is disturbed in the K3 mice. We show that under pathological conditions hyperphosphorylated Tau interacts with c-Jun N-terminal kinase- interacting protein 1 (JIP1), which is associated with the kinesin motor protein complex. As a result, transport of JIP1 into the axon is impaired, causing JIP1 to accumulate in the cell body. Because we found trapping of JIP1 and a pathological Tau/JIP1 interaction also in AD brain, this may have pathomechanistic implications in diseases with a Tau pathology. This is supported by JIP1 sequestration in the cell body of Tau-transfected primary neuronal cultures. The pathological Tau/JIP1 interaction requires phosphorylation of Tau, and Tau competes with the physiological binding of JIP1 to kinesin light chain. Because JIP1 is involved in regulating cargo binding to kinesin motors, our findings may, at least in part, explain how hyperphosphorylated Tau mediates impaired axonal transport in AD and frontotemporal dementia.The microtubule-associated protein Tau is predominantly found in the axonal compartment of neurons, where it binds to microtubules (1). In human brain, six isoforms of Tau are expressed, due to alternative splicing of exons 2, 3 and 10 (2). Tau consists of an amino-terminal projection domain followed by 3 or 4 microtubule binding repeats (3R or 4R), due to splicing of exon 10, and a carboxyl-terminal tail region. In the AD³ and FTD brain, Tau forms filamentous inclusions (3). They are found in nerve cell bodies and apical dendrites as neurofibrillary tangles (NFTs), in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some amyloid plaques (neuritic plaques) (3). Hyperphosphorylation of Tau is thought to be an initiating step (4), as it detaches Tau from microtubules and makes it prone to form aggregates (1, 5). Whereas in AD no mutations have been identified in the MAPT gene encoding Tau, so far 42 intronic and exonic mutations have been found in familial forms of FTD (6). Their identification assisted in the generation of transgenic mouse models that reproduce NFT formation and memory impairment (7).The models were also instrumental in testing hypotheses that had been brought forward to link Tau pathology to functional impairment (8–10). In particular, defects in axonal transport have been implicated in neurodegenerative disorders (11, 12). Tau binding to microtubules affects axonal transport (13), and in cell culture overexpression of Tau was shown to lead to impaired transport of mitochondria and vesicles (14, 15). Axonal transport defects have also been reproduced in wild-type Tau transgenic mice (16) and in K369I mutant Tau K3 mice (17), whereas Tau expression failed to inhibit axonal transport in other systems (18, 19). This apparent discrepancy may depend on the type of cargos analyzed and, specifically, the experimental paradigm, e.g. using phosphorylated (16, 17, 20) versus non-phosphorylated Tau (18).To dissect Tau-mediated axonal transport defects at a molecular level, we used K3 mice that overexpress human Tau carrying the pathogenic FTD K369I mutation (17). We observed a pronounced hyperphosphorylation of transgenic Tau in many brain areas. Clinically, the mice present with an early onset motor phenotype that is, at least in part, caused by impairment of axonal transport in neurons of the substantia nigra. Interestingly, only selected aspects of anterograde axonal transport were impaired, in particular those of kinesin-I motor complex-driven vesicles and mitochondria. Our data suggest a selective impairment of axonal transport rather than a generalized, non-selective blockage of microtubules that has been established in cell culture systems, which fail to phosphorylate Tau at the high levels that are found in vivo even under physiological conditions. More importantly, in AD and FTD Tau is even more phosphorylated, i.e. hyperphosphorylated at physiological sites and de novo at pathological sites, preventing it from binding to microtubules (1).Based on our findings of an impaired kinesin-I-driven axonal transport in the K3 mice, we speculated that hyperphosphorylated Tau may impair anterograde transport by interfering directly with components of the kinesin-I motor complex rather than disrupting the binding of the kinesin heavy chain (see below) to microtubules. Axonal transport along microtubules is mediated by members of the kinesin superfamily (KIF) of motor proteins (21–23). The KIFs typically consist of an ATPase domain that interacts with microtubules and drives movement and a domain that links to cargos, either directly or indirectly, as in the case of KIF5, by assembling with the kinesin light chain (KLC) to form the kinesin-I (KIF5/KLC) motor complex (24). In addition, increasing evidence suggests that scaffolding proteins mediate and regulate the binding of cargos to KIFs (21, 25–27). These include the scaffold protein JNK-interacting protein (JIP) that is involved in the linkage of cargos to the kinesin-I motor complex via KLC (25, 28–33).Here, by using the K3 mouse model, we identified a novel interaction of Tau and JIP in neurons that causes a trapping of JNK interacting protein 1 (JIP1) in the cell body of K3 mice, cell culture systems, and human AD brain. We found that the pathological interaction of hyperphosphorylated Tau and JIP1 competes with the physiological binding of JIP1 to KLC.     

海鲈核糖体蛋白L8cDNA的克隆与进化分析

本文构建了海鲈(Lateolabrax japonicus)头肾全长eDNA文库.PCR方法扩增得到海鲈的核糖体蛋白L8基因,全长848bp,编码257个氨基酸,含有L2及L2-C两个保守区.进化分析结果表明,以L8为参照的进化鉴定结果同经典的分子生物学标准18s鉴定结果十分相似,因此核糖体蛋白L8基因L8可以作为鉴定物种进化程度的新标准.     

Unbiased Selective Isolation of Protein N-terminal Peptides from Complex Proteome Samples Using Phospho Tagging (PTAG) and TiO2-based Depletion

A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products.     

核糖体蛋白L6/Taxreb107的核定位信号的分析

核糖体蛋白L6（RpL6,Taxreb107）含有三个具有核定位信号特征的基序.用作者构建的核定位信号捕获系统分析了这些核定位信号是否具有介导蛋白质进行核转位的功能.将RpL6/Taxreb107分段插入核定位信号捕获载体的克隆位点后转化宿主酵母,发现其前两个核定位信号可以介导融合蛋白进入细胞核,而第三个核定位信号无此作用.将RpL6/Taxreb107分段与绿色荧光蛋白融合后转染培养的哺乳类细胞,证实了以上在酵母中所得的结果.进一步发现RpL6/Taxreb107的前两个核定位信号同时具有核仁定位的功能.当在细胞中表达的早期,进入核内的融合蛋白优先定位于核仁.这些结果一方面有助于理解RpL6/Taxreb107核转位的机理,同时说明作者构建的核定位信号捕获系统也可用在蛋白质中寻找核定位信号.     

Regulation of Protein Kinase A Activity by p90 Ribosomal S6 Kinase 1

Previously, we reported that the catalytic subunit of cAMP-dependent protein kinase (PKAc) binds to the active p90 ribosomal S6 kinase 1 (RSK1) (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell. Biol. 26, 4586–4600). Herein, by overexpressing hemagglutinin-tagged RSK1 fragments in HeLa cells we have identified the region of RSK1 that is responsible for the interaction with PKAc. PKAc bound to the last 13 amino acids of RSK1, which overlaps the Erk1/2 docking site. This interaction between PKAc and RSK1 required the phosphorylation of Ser-732 in the C terminus of RSK1. Depending upon its phosphorylation status, RSK1 switched interactions between Erk1/2 and PKAc. In addition, a peptide corresponding to the last 13 amino acids of RSK1 with substitution of Ser-732 with Glu (peptide E), but not Ala (peptide A), decreased interactions between endogenous active RSK1 and PKAc. RSK1 attenuated the ability of cAMP to activate PKA in vitro and this modulation was abrogated by peptide E, but not by peptide A. Similarly, in intact cells, cAMP-mediated phosphorylation of Bcl-xL/Bcl-2-associated death promoter on Ser-115, the PKA site, was reduced when RSK1 was activated by epidermal growth factor, and this effect was blocked by peptide E, but not by peptide A. These findings demonstrate that interactions between endogenous RSK1 and PKAc in intact cells regulate the ability of cAMP to activate PKA and identify a novel mechanism by which PKA activity is regulated by the Erk1/2 pathway.     

Ribosomal protein interactions in yeast. Protein L15 forms a complex with the acidic proteins

Protein L15 from Saccharomyces cerevisiae ribosomes has been shown to interact in solution with acidic ribosomal proteins L44, L44' and L45 by different methods. Thus, the presence of the acidic proteins changes the elution characteristics of protein L15 from CM-cellulose and DEAE-cellulose columns and from reverse-phase HPLC columns. Moreover, immunoprecipitation using anti-L15 specific monoclonal antibodies coprecipitates the acidic proteins, too. Conversely, antibodies raised against the acidic proteins immunoprecipitate protein L15. This coprecipitation seems to be specific since it does not involve other ribosomal proteins present in the sample. Similarly, plastic-adsorbed antibodies specific for one of the components in the L15--acidic-protein complex are able to retain the other component of the complex but cannot bind unrelated proteins. Moreover, protein L15 can be chemically cross-linked to the acidic proteins in solution. These results indicate that protein L15 might be equivalent to bacterial ribosomal protein L10 in forming a complex with the acidic proteins. Since, on the other hand, protein L15 has been shown to be immunologically related to bacterial protein L11 [Juan Vidales et al. (1983) Eur. J. Biochem. 136, 276-281] and to interact with the same region of the large ribosomal RNA as does protein L11 [El-Baradi et al. (1987) J. Mol. Biol. 195, 909-917], these results suggest strongly that protein L15 plays the same role in the yeast ribosome as proteins L10 and L11 do in the bacterial particles.     

Differential Effects of Replacing Escherichia coli Ribosomal Protein L27 with Its Homologue from Aquifex aeolicus

The rpmA gene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophile Aquifex aeolicus, and the protein was overexpressed and purified. Comparison of the A. aeolicus protein with its homologue from Escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the E. coli protein is unstructured under the same conditions. A mutant of E. coli that lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression of E. coli L27 from an extrachromosomal copy of the rpmA gene. When A. aeolicus L27 was expressed in the same mutant, an increase in the growth rate occurred and the "foreign" L27 protein was incorporated into E. coli ribosomes. However, the presence of A. aeolicus L27 did not promote 50S subunit assembly. Thus, while the A. aeolicus protein can apparently replace its E. coli homologue functionally in completed ribosomes, it does not assist in the assembly of E. coli ribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.     

Molecular Requirements for T Cell Recognition of N-Myristoylated Peptides Derived from the Simian Immunodeficiency Virus Nef Protein

We have recently isolated a rhesus macaque cytotoxic T cell line, 2N5.1, that specifically recognizes an N-myristoylated 5-mer peptide (C₁₄-Gly-Gly-Ala-Ile-Ser [C14nef5]) derived from the simian immunodeficiency virus (SIV) Nef protein. Such C14nef5-specific T cells expand in the circulation of SIV-infected monkeys, underscoring the capacity of T cells to recognize viral lipopeptides; however, the molecular basis for the lipopeptide antigen presentation remains to be elucidated. Here, functional studies indicated that the putative antigen-presenting molecule for 2N5.1 was likely to have two separate antigen-binding sites, one for interaction with a C₁₄-saturated acyl chain and the other for anchorage of the C-terminal serine residue. Mutants with alanine substitutions for the second glycine residue and the fourth isoleucine residue were not recognized by 2N5.1 but interfered with the presentation of C14nef5 to 2N5.1, indicating that these structural analogues retained the ability to interact with the antigen-presenting molecules. In contrast to the highly specific recognition of C14nef5 by 2N5.1, an additional cytotoxic T cell line, SN45, established independently from a C14nef5-stimulated T cell culture, showed superb reactivity to both C14nef5 and an N-myristoylated Nef 4-mer peptide, and therefore, the C-terminal serine residue was dispensable for the recognition of lipopeptides by the SN45 T cells. Furthermore, the mutants with alanine substitutions were indeed recognized by the SN45 T cells. Given that N-myristoylation of the Nef protein occurs in the conserved motifs and is critical for viral pathogenesis, these observations predict that the lipopeptide-specific T cell response is difficult for viruses to avoid by simply introducing amino acid mutations.     

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H₂O₂ concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.     

Antibody Constant Region Peptides Can Display Immunomodulatory Activity through Activation of the Dectin-1 Signalling Pathway

We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG(1), is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.