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Bone defects are a significant health problem worldwide, as bone is the second-most transplanted tissue after blood. Although a myriad of bone grafts (BGs) have been used to treat bone repairs, none of them possesses all the desirable characteristics. An approach to improve BGs is to add bio-active components, however often difficult as BG production may disrupt the biological activities of such molecules. Here, we present a composite xenohybrid BG, SmartBonePep, with a type of biomolecule inspired by intrinsically disordered proteins (IDPs). These synthetic peptides (named P2 and P6) are physically entrapped into the polymer matrix of the composite BG. The effects of SmartBonePep on human osteoblasts were tested. Results showed that SmartBonePep enhanced proliferation and osteogenic effects. In order to verify the bioactivity of P2 and P6, these peptides were tested indirectly by being added to cell culture media too. Here, P2 or P6 exhibited promoting effects on osteogenic-related gene expressions. In this study, we showed highly effective osteoinductive synthetic peptides P2 or P6, which possess proline-rich and intrinsically disordered structural characters. This use of IDPs may provide promising bone enhancement biomolecules for clinical usage.

Proline rich proteins (PRPs), earlier famous as animal salivary proteins, have now been proven as indispensable plant proteins. They are highly rich in proline amino acid residues at the N-terminus whereas a characteristic eight cysteine motif is located at the C-terminus. The PRPs support a number of developmental processes from germination to plant death. Under normal environmental conditions, PRP genes express customarily in different plant parts depending on the specific function to be carried out. During abiotic stresses, PRP genes exhibit an uneven pattern of transcriptional regulation depending on the time and intensity of stress. Transgenic plants overexpressing PRP genes show an enhanced tolerance to abiotic stresses. This review focuses on contemporary functions of PRPs during stresses and proposes that PRPs are involved in the regulation of free cellular proline content during stress in a well synchronized manner.

Proline-rich macrocyclic peptides (PRMPs) are natural products present in geographically and phylogenetically dispersed marine sponges. The large diversity and low abundance of PRMPs in sponge metabolomes precludes isolation and structure elucidation of each individual PRMP congener. Here, using standards developed via biomimetic enzymatic synthesis of PRMPs, a mass spectrometry-based workflow to sequence PRMPs was developed and validated to reveal that the diversity of PRMPs in marine sponges is much greater than that has been realized by natural product isolation-based strategies. Findings are placed in the context of diversity-oriented transamidative macrocyclization of peptide substrates in sponge holobionts.

Background. Saliva, as a complex biofluid, plays a pivotal role in maintaining oral health and tooth integrity. There has been inconsistent data available on the relationship between salivary parameters and oral health. This study aims to investigate the association between salivary statherin, acidic proline-rich proteins (aPRP), and calcium with oral hygiene status. Methods. One hundred and eighty-eight healthy subjects aged between 18 and 50 years with varying oral hygiene status who gave consent to participate were included in this cross-sectional study. The subjects were recruited from primary oral health care of MAHSA University. Oral hygiene of all the participants was measured using Oral Hygiene Index–Simplified (OHI-S). Stimulated saliva collected using paraffin wax was analyzed for salivary statherin, aPRP, and calcium. The relationship between salivary statherin, aPRP, and calcium levels with OHI-S was assessed using Spearman’s Rank correlation coefficient; the strength of relationship was assessed by multiple linear regression analysis. Results. The study found a weak positive correlation (r = 0.179, p = 0.014) between salivary statherin and OHI-S; weak negative correlation (r = −0.187, p = 0.010) between salivary aPRP and OHI-S; and moderate negative correlation between salivary statherin and salivary aPRP levels (r = −0.50, p

Background and Aims: Cardiac injury induces dynamic changes in the expression of microRNAs (miRs). For example, the evolutionarily conserved miR-150 is downregulated in patients with multiple cardiovascular diseases such as myocardial infarction (MI) and cardiomyopathies, as well as in various mouse models of heart failure (HF). MiR-150 is significantly associated with HF severity and outcome in humans. Using a systemic miR-150 knockout (KO) mouse model, we previously showed that carvedilol (Carv)/β 1 -adrenergic receptor/β-arrestin1-responsive miR-150 confers cardiac protection against MI (Left side in Figure). However, the extent to which expression of miR-150 in cardiomyocytes (CMs) regulates MI is unknown and there is a lack of mechanistic insight by which CM miR-150 modulates cardiac protection. Methods and Results: Here, we demonstrate using a novel mouse model that conditional CM-specific miR-150 KO (miR-150 cKO) in mice worsens cardiac dysfunction, stress, fibrosis and apoptosis post-MI, without affecting mortality or inflammation. Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline-rich protein 1a (sprr1a) as a novel regulatory target of miR-150. Our mouse and CM studies further reveal that sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion. In contrast, its expression is downregulated in hearts and CMs by Carv. Our human heart data also show that left ventricular sprr1a is upregulated in patients with HF with reduced ejection fraction. Mechanistically, the cardioprotective roles of CM miR-150 during MI are in part attributed to the direct and functional repression of pro-apoptotic gene sprr1a in CMs (Right side in Figure). Conclusions: These findings reveal a pivotal role for the miR-150/sprr1a axis in regulating CM function post-MI, and this novel axis could be a therapeutic target for intervention in ischemic heart disease.

Croissamide, a proline-rich cyclic peptide that contains an N-prenylated tryptophan, was isolated from a marine cyanobacterium Symploca sp. Its gross structure was determined by spectroscopic analyses, and the absolute configuration was established based on chiral HPLC analyses of acid hydrolysates.

A new proline-rich antimicrobial peptide from alpaca (Vicugnia pacos) was obtained, as well as its modified analogs. The biological activity of the peptides was studied, and also the role of the N- and C-terminal fragments, when acting on bacterial cells, was analyzed.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance system that maintains the integrity of transcripts by removing aberrant mRNAs harbouring premature termination codons (PTCs). In the absence of NMD, truncated proteins with dominant negative or potentially deleterious gain of function activity may be expressed from these erroneous mRNAs. In addition to its function as a quality control for mRNAs, NMD is involved in the regulation of 3 – 10% of the Saccharomyces cerevisiae, Drosophilia melanogaster, and Homo sapiens transcriptomes. PTC recognition in NMD requires the cross talk between terminating ribosome stalled at stop codon and downstream cis-acting signal that is not conserved across species. In S. cerevisiae and D. melanogaster, the faux 3’ untranslated region (UTR) model has been proposed to be the main mechanism in recognising PTC. In mammals, PTC recognition is mediated through the interaction of the SMG1, Upf1, eukaryotic Release factor 1 and 3 (SURF) complex on the terminating ribosome at a PTC and downstream exon junction complex (EJC) during the pioneer round of translation. Upon recognition of PTC, an evolutionarily conserved surveillance complex consisting of the Upf1, Upf2 and Upf3 proteins is assembled. In mammals, the assembly of the surveillance complex also activates a phosphoinositide-3-kinase related protein kinase, SMG1, which will phosphorylate the Upf1 protein. Till date, three mechanisms of how phosphorylated Upf1 protein leads to decay of mRNAs have been proposed. First, the phosphorylated Upf1 protein recruits the SMG7 protein and C terminal of SMG7 protein targets the associated mRNA for decay through an undetermined mechanism. Second, the phosphorylated Upf1 protein recruitshDcp1a or Dcp2 proteins to decap mRNA for its subsequent 5’-3’ decay. Third, the hyperphosphorylated Upf1 protein associates with the nuclear receptor coregulatory protein 2 (PNRC2), which targets the mRNA to the P body through the interaction between the PNRC2 and hDcp1a proteins. Subsequently, decapping and 5’ - 3’ decay of mRNA occurs in the P body. As the three mechanisms are inferred from the results of coimmunoprecipitation or two-hybrid interactions, no direct interaction between the hyperphosphorylated Upf1 protein and decapping enzymes has been demonstrated till date. Here we present the crystal structure of hDcp1a protein in complex with the PNRC2 peptide. The proline-rich region of the PNRC2 peptide is bound to the EVH1 domain of the hDcp1a protein while isothermal titration calorimetry study demonstrates that NR-box of the PNRC2 protein mediates the direct interaction with hyperphosphorylated Upf1 protein. The mode of the PNRC2 protein interaction with the hDcp1a protein is distinct from those observed in other classes of EVH1/proline-rich ligands interactions. Additionally, PNRC2 mutagenesis study performed by our collaborator showed that disruption of the interaction of the PNRC2 protein with the hDcp1a protein abolishes its P-body localization and ability to promote mRNA degradation when tethered to mRNAs. Furthermore, the PNRC2 protein acts in synergy with the hDcp1a protein to stimulate the decapping activity of the Dcp2 protein by bridging the interaction between the hDcp1a and Dcp2 proteins. The formation of a novel PNRC2-hDcp1a-Dcp2 decapping complex suggests that the PNRC2 protein is a decapping coactivator in addition to its adaptor role in NMD. DOCTOR OF PHILOSOPHY (SBS)

The interaction between CrkII and cAbl is implicated in diverse cellular processes. This interaction starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to the proline-rich motifs of cAbl (PRMscAbl). Despite its critical importance, the detailed binding mechanism between the nSH3 domain and PRMs remains elusive. In this study, we used nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiment to study the binding kinetics between the nSH3 domain of CrkII and PRMscAbl. Our results highlight that the nSH3 domain binds to three PRMscAbl with very high on- and off-rate constants, indicating the transient nature of the binding. To further characterize the binding transition state, we conducted the Eyring and linear free energy relationship analyses using temperature-dependent kinetic data. These data indicate that the binding transition state of the nSH3 domain and PRM is accompanied by small activation enthalpy, owing to partial desolvation of the transition state. These results also highlight the similarity between the transition and free states, in terms of structure and energetics. Although the binding of the nSH3 domain and PRM displays the features consistent with a diffusion-limited process within our experimental conditions, further tests are necessary to determine if the binding is a true diffusion-limited process.

Conformational heterogeneity is critical to understanding protein function but challenging to quantify. Experimental approaches that can provide sufficient temporal and spatial resolution to measure even rapidly interconverting states at specific locations in proteins are needed to fully elucidate the contribution of conformational heterogeneity and dynamics to function. Infrared spectroscopy in combination with the introduction of carbon deuterium bonds, which provide frequency-resolved probes of their environments, can uncover rapidly interconverting states with residue-specific detail. Using this approach, we quantify conformational heterogeneity of proline-rich peptides associated with different proline backbone conformations, as well as reveal their dependence on the sequence context.

Pneumococcal surface protein A (PspA) is a surface exposed, highly immunogenic protein of Streptococcus pneumoniae. Its N-terminal α-helical domain (αHD) elicits protective antibody in humans and animals that can protect mice from fatal infections with pneumococci and can be detected in vitro with opsonophagocytosis assays. The proline-rich domain (PRD) in the center of the PspA sequence can also elicit protection. This study revealed that although the sequence of PRD was diverse, PRD from different pneumococcal isolates contained many shared elements. The inferred amino acid sequences of 123 such PRDs, which were analyzed by assembly and alignment-free (AAF) approaches, formed three PRD groups. Of these sequences, 45 were classified as Group 1, 19 were classified as Group 2, and 59 were classified as Group 3. All Group 3 sequences contained a highly conserved 22-amino acid non-proline block (NPB). A significant polymorphism was observed, however, at a single amino acid position within NPB. Each of the three PRD groups had characteristic patterns of short amino acid repeats, with most of the repeats being found in more than one PRD group. One of these repeats, PKPEQP as well as the NPB were previously shown to elicit protective antibodies in mice. In this study, we found that sera from 12 healthy human adult volunteers contained antibodies to all three PRD groups. This suggested that a PspA-containing vaccine containing carefully selected PRDs and αHDs could redundantly cover the known diversity of PspA. Such an approach might reduce the chances of PspA variants escaping a PspA vaccine's immunity.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. SS new components of the family were characterized by top-down liquid chromatography mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S-1 -> A, P-Ko P-36 -> S, and P-Ko A(41) -> S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

Proline-rich protein 11 (PRR11) has been shown to play an critical roles in the development of cancer. However, the clinical significance and the biological role of PRR11 in hepatocellular carcinoma (HCC) remains unknown. The present study aimed to investigate the expression pattern, prognostic value and the biological role of PRR11 in HCC. PRR11 expression in 80 HCC surgical specimens was examined, and its clinical significance was analyzed. The role of PRR11 in cell proliferation, colony formation, migration and invasion were also determined. The results showed that PRR11 mRNA was significantly up-regulated in 56.25% (45/80) HCC from that in matched adjacent non-tumor tissues. High PRR11 was correlated with tumor size (P = 0.01) and TNM stage (P = 0.006). Patients with higher PRR11 expression had poor overall survival time (P 0.001). Furthermore, PRR11 silencing obviously inhibited cell proliferation, colony formation, as well as cell migration and invasion of HCC cell lines in vitro. Mechanistically, knockdown of PRR11 significantly decreased the expression of β-catenin, cyclinD1, c-myc and N-cadherin in HCC cell lines. Additionally, the inhibitory effects of PRR11 silencing on cell migration was significantly enhanced by β-catenin inhibition. PRRl1 mRNA expression was found positively correlated with β-catenin (R = 0.5472, P ˂ 0.0001), c-myc (R = 0.5527, P ˂ 0.0001) and cyclinD1 (R = 0.3948, P = 0.0003) in HCC tissues. Collectively, our data demonstrate that PRR11 plays an oncogenic role in HCC progression, through activating the Wnt/β-catenin signaling pathway, and may represent a valuable prognostic marker and therapeutic target for HCC.

Mutations in the primate-specific proline-rich domain (PRD) of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are thought to cause Leber congenital amaurosis or dominant cone-rod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL11-316 with PRD truncation, and the P351Δ12 and P376S mutants were examined by small angle X-ray scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506-binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecule's radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase-6A and HSP90. We also identified the γ-subunit of phosphodiesterase-6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351Δ12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants. Mutations in the proline-rich domain (PRD) of human AIPL1 cause severe retinal diseases, yet the role of PRD and the mechanisms of PRD mutations are unknown. Here, we describe a SAXS-derived solution structure of AIPL1 and functional properties of disease-linked AIPL1-PRD mutants. This structure and functional analyses provide a framework for understanding the mechanisms of PRD in disease.

The naturally occurring proline-rich cyclic octapeptide stylissamide X is for the first time total synthesized in the present study by a two-step solid-phase/solution synthesis strategy using two different synthetic routes. In this study, all the amino acids are of L-configuration. The linear octapeptide was assembled by standard Fmoc solid-phase peptide synthesis (SPPS) and cyclization was carried out subsequently by the solution method. The target compound was purified by preparative RP-HPLC, and its structure was identified by 13C NMR, 1H NMR and MS.

In this study, we have evaluated by HPLC-DAD, DLS and MALDI-TOF a synergic effect of the coexistence of two salivary-PRP fractions (basic-PRPs and acidic PRPs) on the interaction with flavanols. Results obtained showed noticeable enhancement of the interaction between (epi)catechin and PRPs when both types of proteins are blended. Up to 30 soluble aggregates have been tentatively identified with molecular weight from 4680 to 35,851. (epi)Catechins seem to bind preferentially bPRPs than aPRPs, although the medium size aggregates flavanol-bPRPs formed could favour the interaction with aPRPs giving rise to soluble mixed aggregates.

Dental caries is a chronic infectious disease that affects billions of people with large individual differences in activity. We investigated whether PRH1 and PRH2 polymorphisms in saliva acidic proline-rich protein (PRP) receptors for indigenous bacteria match and predict individual differences in the development of caries. PRH1 and PRH2 variation and adhesion of indigenous and cariogenic (Streptococcus mutans) model bacteria were measured in 452 12-year-old Swedish children along with traditional risk factors and related to caries at baseline and after 5-years. The children grouped into low-to-moderate and high susceptibility phenotypes for caries based on allelic PRH1, PRH2 variation. The low-to-moderate susceptibility children (P1 and P4a−) experienced caries from eating sugar or bad oral hygiene or infection by S. mutans. The high susceptibility P4a (Db, PIF, PRP12) children had more caries despite receiving extra prevention and irrespective of eating sugar or bad oral hygiene or S. mutans-infection. They instead developed 3.9-fold more caries than P1 children from plaque accumulation in general when treated with orthodontic multibrackets; and had basic PRP polymorphisms and low DMBT1-mediated S. mutans adhesion as additional susceptibility traits. The present findings thus suggest genetic autoimmune-like (P4a) and traditional life style (P1) caries, providing a rationale for individualized oral care. Wos title: eGenetic- and Lifestyle-dependent Dental Caries Defined by the Acidic Proline-rich Protein Genes PRH1 and PRH2

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1–35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa . By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1–35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli . The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

The application of colloidal particles from natural materials for purposes ranging from the delivery of bioactives to interfacial stabilisation and bulk structuring have recently gained a lot of interest for applications in the field of fast moving consumer goods, nutraceuticals, agricultural formulations and medicine. Zein-a proline rich water insoluble protein obtained from natural and sustainable source has been recently researched to generate colloidal structures that can find a wide range of applications. In this paper, we review the recent progress in the preparation of colloidal structures and their further application as functional materials in the field of delivery of functional ingredients and structuring of bulk phases and interfaces.

Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family. We used Pyk2 knockout (Pyk2-KO) mice to study the role of Pyk2 in cutaneous wound repair. We report that the rate of wound closure was delayed in Pyk2-KO compared with control mice. To examine whether impaired wound healing of Pyk2-KO mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-KO and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair was decreased in Pyk2-KO keratinocytes compared with WT cells. Moreover, cultured human epidermal keratinocytes overexpressing the dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure and was associated with increased expression of matrix metalloproteinase (MMP)-1, MMP-9, and MMP-10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by coexpression of the dominant-negative mutant of PKCδ and by GM-6001, a broad-spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCδ and MMP functions.

Epithelial tissues are specialized to protect underlying tissues from environmental influences such as physical and chemical agents, infection by invasive microorganisms as well as water and heat loss. They are grouped into simple, transitional and stratified epithelia, which line the cavities and surfaces of structures throughout the body, and also form glands, separate compartments, regulate the exchange of molecules and act as sensory organs. Stratified epithelia such as the epidermis and the gingival and hard palate mucosa are in constant renewal, with cells proliferating in the lower layers, while the intermediate stratum and outermost layers undergo a tissue-specific process of differentiation to form a protective cornified barrier. This review focuses on a subclass of structural proteins, the small proline-rich proteins (SPRRs), which constitute cornified cell envelope precursors. Several studies have suggested that the SPRRs are related to increased epithelial proliferation and to malignant processes. Hence, we also review the literature for more extensive and in-depth profile of these proteins in cancer and other diseases. The understanding of SPRR functions has advanced in recent years, but many important questions about their role in pathophysiological processes remain unanswered, which stimulate new studies and approaches.

With the widespread applications of nanomaterials such as carbon nanotubes, there is a growing concern on the biosafety of these engineered nanoparticles, in particular their interactions with proteins. In molecular simulations of nanoparticle-protein interactions, the choice of empirical parameters (force fields) plays a decisive role, and thus is of great importance and should be examined carefully before wider applications. Here we compare three commonly used force fields, CHARMM, OPLSAA, and AMBER in study of the competitive binding of a single wall carbon nanotube (SWCNT) with a native proline-rich motif (PRM) ligand on its target protein SH3 domain, a ubiquitous protein-protein interaction mediator involved in signaling and regulatory pathways. We find that the SWCNT displays a general preference over the PRM in binding with SH3 domain in all the three force fields examined, although the degree of preference can be somewhat different, with the AMBER force field showing the highest preference. The SWCNT prevents the ligand from reaching its native binding pocket by (i) occupying the binding pocket directly, and (ii) binding with the ligand itself and then being trapped together onto some off-sites. The pi-pi stacking interactions between the SWCNT and aromatic residues are found to play a significant role in its binding to the SH3 domain in all the three force fields. Further analyses show that even the SWCNT-ligand binding can also be relatively more stable than the native ligand-protein binding, indicating a serious potential disruption to the protein SH3 function.

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Fil: Gebhard, Leopoldo German. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Iglesias, Nestor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Byk, Laura Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Filomatori, Claudia Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: de Maio, Federico Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Tau that belongs to the family of microtubule-associated proteins is the major constituent of intraneuronal fibrillar lesions described in Alzheimer's Disease (AD). Its sequence homology with salivary proline-rich proteins (sPRP), known to fix tannins, combined with some epidemiological evidences that a moderate consumption of polyphenols presents benefits in AD, suggests that tannins can bind Tau protein and, perhaps, could inhibit or modulate the cascade of events leading to AD symptoms. To study the affinity of tannins towards the Tau proline rich domain P2, we have first synthesized a peptide representative of this domain, determined its 3D-structure and its affinity towards different procyanidins (epicatechin-EC, epicatechin gallate-ECG, epigallocatechin gallate-EGCG and procyanidin B3 and B2) by using both NMR, molecular modelling and dynamic techniques. We have found that the Tau peptide is able to fix the different tested tannins in two distinct domains with an affinity in the mM range.

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the two-fold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.

The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some of its structural analogues was investigated by NMR-spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis'-conformation of Pro3 and the gamma-turn on the Leu4-residue: for Val2 and Phe5 a beta-structure seems to be privileged. In such a conformation Val2 and Leu4 residues occupy exactly the same positions in space as residues i and i + 3 in an alpha-helix. It suggests that the PRP-hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by "knobs into holes" packing.

The primary structures of the four human salivary anionic proline-rich proteins and an analogous protein from the saliva of a monkey (Macaca arctoides) have been further investigated. These proteins possess the unusual property of inhibiting crystal growth of calcium phosphate salts, and it has been proposed that they play an important role in the mouth, by preventing precipitation of calcium phosphate salts from the supersaturated saliva. The tryptic fragments responsible for this activity have been isolated from all five proteins and their complete amino acid sequences determined and compared. These active fragments have been unequivocally identified as the amino-terminal segment in all five proteins. The structures of the four human fragments are identical except for the presence of Asn at residue 4 in PRP-1 and -3 instead of Asp found in PRP-2 and -4. Comparison of the 30 residue human fragments with the monkey fragment shows 18 residues to be identical in these peptides, providing that residue 1 of the monkey fragment is aligned with residue 3 of the human proteins. These studies constitute the next step in determining the mechanism of action of these unusual proteins, and in determining their minimum chain length required for inhibitory activity.

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3–4 days of human BM stromal cell cultivation in the presence of 2–20 μg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3–4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus–bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation.