Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa)

Scope: Today, about 2–8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Methods and results: Sera from 42 Spanish lettuce-allergic patients were obtained from pa-tients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Conclusion: Two new major lettuce allergens—a thaumatin-like protein and an aspartyl protease—have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

Constitutive and regulated α-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease

Amyloid β peptide (Aβ), the principal proteinaceous component of amyloid plaques in brains of Alzheimer’s disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative α-secretase within the Aβ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsα into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated α-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous α-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Aβ fragments confirm the correct α-secretase cleavage site and demonstrate a dependence on the substrate’s conformation. Our results provide evidence that ADAM 10 has α-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer’s disease.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in Escherichia coli and purified. After refolding rPrP into an α-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base–base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSα (hMSH2⋅hMSH6) and hMutSβ (hMSH2⋅hMSH3). In HL-60 extracts the hMutSα to hMutSβ ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base–base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSα and hMutSβ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion–deletion heterology, but only hMutSα restores base–base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition

LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process. Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle. Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids. Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein–protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.

Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Meiosis-specific homologs of RecA protein have been identified in Saccharomyces cerevisiae and higher eukaryotes including mammals, but their enzymatic activities have not been described. We have purified the human protein HsDmc1 produced in Escherichia coli from a cloned copy of the cDNA. The recombinant enzyme had DNA-dependent ATPase activity with an estimated kcat of 1.5 min−1. DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues. HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides. The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATPγS.

Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.

Structural protein 4.1 is located in mammalian centrosomes

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.