In Vitro Identification of Histatin 5 Salivary Complexes

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance.     

Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection

A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, α-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.     

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.     

Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.     

A new method for the isolation of histatins 1, 3, and 5 from parotid secretion using zinc precipitation

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.     

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.     

Mapping protein receptor-ligand interactions via in vivo chemical crosslinking, affinity purification, and differential mass spectrometry

Protein receptor-ligand interactions play important roles in mediating enzyme catalysis, signal transduction, and other protein functions. Immunoaffinity purification followed by mass spectrometry analysis is a common method for identifying protein receptor-ligand complexes. However, it is difficult to distinguish between specific protein binding partners and non-specifically bound proteins that co-purify with the complex. In addition, weakly interacting binding partners may dissociate from the protein receptor-ligand complexes during immunoaffinity purification. The combination of chemical crosslinking, affinity purification, and differential mass spectrometry analysis provides a direct method for capturing stable, weak, and transient protein interactions that occur in vivo and in vitro. This approach enables the identification of functional receptor-ligand binding partners with high confidence. Herein, we describe a differential mass spectrometry approach coupled with in situ chemical crosslinking and immunoaffinity purification for identifying receptor-ligand binding partners. In particular, we identified a functional, counter-ligand structure of the natural killer cell p30-related protein.     

Zinc and copper bind to unique sites of histatin 5

Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C(epsilon1)-H histidyl and the C(gamma)-H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C(beta)-H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the -H-E-X-X-H- zinc binding motif, and copper binding occurs within the N-terminal D-S-H-, ATCUN motif.     

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

Death-associated protein kinase 2 (DAPK2) is a Ca²⁺/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein''s subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.     

蚜虫唾液蛋白研究进展

蚜虫属于半翅目蚜科,多为重要的农业害虫,通过刺吸式口器吸食植物汁液,传播病毒,其爆发常常造成重大经济损失。在漫长的协同进化历程中,植物建立了高效的防御系统以应对蚜虫威胁。为了克服植物的防御反应,蚜虫也发展了相应的反制手段,其中蚜虫在取食过程中分泌的唾液蛋白能调控植物防御反应,降解植物次生物质,从而在蚜虫与植物互作中发挥着至关重要的作用。本文综述了蚜虫唾液蛋白的组分鉴定方法和相关蛋白的功能,并对唾液蛋白在蚜虫防治的应用和今后的研究方向进行了展望。常见的蚜虫唾液蛋白组分的鉴定和分析方法包括唾液蛋白的酶活性分析、唾液蛋白组学分析、唾液腺转录组学和蛋白组学分析等。但这些方法各有利弊,仅采取一种分析方法不能客观全面地反映蚜虫唾液蛋白分泌谱,多种技术手段联合分析方可提供更为逼真详实的信息。蚜虫唾液蛋白种类繁多,可分为解毒酶、保护酶、水解酶、结合功能蛋白以及分类未知的效应蛋白等。蚜虫唾液蛋白功能多样,能参与唾液鞘的形成,诱导植物防御反应,促进蚜虫取食,提高蚜虫繁殖力等。通过RNAi干扰唾液蛋白编码基因会显著改变蚜虫取食行为,并降低蚜虫存活率、产蚜量和适合度。因此,唾液蛋白是防控蚜虫的理想靶标。目前,采用寄主诱导的基因沉默(host-induced gene silencing, HIGS)技术已培育了数种靶向唾液蛋白基因的高效抗蚜作物品系,展示出了良好的应用前景。从目前研究来看,各种蚜虫唾液蛋白谱急需采用多组学手段联合分析的方法来进行完整解析。各种唾液蛋白的具体功能方面的研究还严重缺乏,需从蚜虫、植物、两者之间的互作等多维度探究唾液蛋白的作用及相关的分子机制,为发展基于蚜虫唾液蛋白调控的蚜虫防治新策略打下基础。     

Biochemical and computational analysis of LNX1 interacting proteins

PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.     

Proteomic Evaluation of Acquired Enamel Pellicle during In Vivo Formation

Acquired enamel pellicle (AEP) is a protein film that forms on the enamel surface of teeth by selective adsorption of proteins and peptides present in the mouth. This protein film forms the interface between enamel and the damage oral biofilm, which modulates the attachment of bacteria found in oral biofilm. The overall goal of this study was to gain insight into the biological formation of the human in vivo AEP. This study hypothesized that AEP is created by the formation of successive protein layers, which consist of initial binding to enamel and subsequent protein-protein interactions. This hypothesis was examined by observing quantitative and qualitative changes in pellicle composition during the first two hours of AEP formation in the oral cavity. Quantitative mass spectrometry approaches were used to generate an AEP protein profile for each time-point studied. Relative proteomic quantification was carried out for the 50 proteins observed in all four time-points. Notably, the abundance of important salivary proteins, such as histatin 1, decrease with increasing of the AEP formation, while other essential proteins such as statherin showed constant relative abundance in all time-points. In summary, this is the first study that investigates the dynamic process to the AEP formation by using proteomic approaches. Our data demonstrated that there are significant qualitative and quantitative proteome changes during the AEP formation, which in turn will likely impact the development of oral biofilms.     

Hidden partners: Using cross‐docking calculations to predict binding sites for proteins with multiple interactions

Protein‐protein interactions control a large range of biological processes and their identification is essential to understand the underlying biological mechanisms. To complement experimental approaches, in silico methods are available to investigate protein‐protein interactions. Cross‐docking methods, in particular, can be used to predict protein binding sites. However, proteins can interact with numerous partners and can present multiple binding sites on their surface, which may alter the binding site prediction quality. We evaluate the binding site predictions obtained using complete cross‐docking simulations of 358 proteins with 2 different scoring schemes accounting for multiple binding sites. Despite overall good binding site prediction performances, 68 cases were still associated with very low prediction quality, presenting individual area under the specificity‐sensitivity ROC curve (AUC) values below the random AUC threshold of 0.5, since cross‐docking calculations can lead to the identification of alternate protein binding sites (that are different from the reference experimental sites). For the large majority of these proteins, we show that the predicted alternate binding sites correspond to interaction sites with hidden partners, that is, partners not included in the original cross‐docking dataset. Among those new partners, we find proteins, but also nucleic acid molecules. Finally, for proteins with multiple binding sites on their surface, we investigated the structural determinants associated with the binding sites the most targeted by the docking partners.     

Interactions between Arabidopsis acyl-CoA-binding proteins and their protein partners

Protein–protein interactions are at the core of cellular interactomics and are essential for various biological functions. Since proteins commonly function as macromolecular complexes, it is important to identify their interacting partners to better understand their function and the significance in these interactions. The acyl-CoA-binding proteins (ACBPs) of eukaryotes show conservation in the presence of a lipid-binding acyl-CoA-binding domain. In Arabidopsis thaliana, four of six members from the AtACBP family possess ankyrin repeats (AtACBP1 and AtACBP2) or kelch motifs (AtACBP4 and AtACBP5), which can potentially mediate protein–protein interactions. Through yeast two-hybrid screens, a dozen putative protein partners interacting with AtACBPs have been isolated from an Arabidopsis cDNA library. Investigations in the past decade on the interaction between AtACBPs and their protein partners have revealed novel roles for AtACBPs, including functions in mediating oxidative stress responses, heavy metal tolerance and oxygen sensing. Recent progress and current questions on AtACBPs and their interactors are discussed in this review.     

Anti-Candidal Activity of Genetically Engineered Histatin Variants with Multiple Functional Domains

The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.     

Poxvirus Proteomics and Virus-Host Protein Interactions

Summary: Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.     

The diverse biofunctions of LIM domain proteins: determined by subcellular localization and protein-protein interaction

The LIM domain is a cysteine- and histidine-rich motif that has been proposed to direct protein-protein interactions. A diverse group of proteins containing LIM domains have been identified, which display various functions including gene regulation and cell fate determination, tumour formation and cytoskeleton organization. LIM domain proteins are distributed in both the nucleus and the cytoplasm, and they exert their functions through interactions with various protein partners.     

Protein-dependent ribozymes report molecular interactions in real time

Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.