Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

Integrative X-ray Structure and Molecular Modeling for the Rationalization of Procaspase-8 Inhibitor Potency and Selectivity.

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ X-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

Understanding lipopolysaccharide aggregation and its influence on activation of Factor C.

The quantification of lipopolysaccharide (LPS) shed by bacteria within aqueous samples is typically performed by binding LPS to a protein called Factor C within a lysate prepared from the blood of horseshoe crabs (Limulus amebocyte lysate (LAL)). How the state of aggregation of LPS impacts Factor C activation, however, is not understood, particularly in the presence of select salts and non-ionic surfactants that are commonly incorporated into pharmaceutical formulations. To address this open question, herein we report on the aggregation status of LPS in aqueous solution, characterized using angle-dependent static and dynamic light scattering with and without chelating salts and polysorbate surfactants, and its correlation with activation of Factor C. Because the aggregation status of LPS is kinetically controlled, care was taken to compare LPS aggregation and activity using identically prepared samples. By plotting LPS activity versus the LPS aggregate size distribution over varied solution conditions, we found a positive correlation between LPS aggregate sizes between 30 and 50 nm and LAL activity. Overall, our results support the hypothesis that activation of Factor C is dependent of LPS aggregate size, and that the modulating effects of salts and surfactants on activation of Factor C is associated with changes in the LPS aggregation.

Molecular characterization of prophenoloxidase-1 (PPO1) and the inhibitory effect of kojic acid on phenoloxidase (PO) activity and on the development of Zeugodacus tau (Walker) (Diptera: Tephritidae).

Phenoloxidase (PO) plays a key role in melanin biosynthesis during insect development. Here, we isolated the 2310-bp full-length cDNA of PPO1 from Zeugodacus tau, a destructive horticultural pest. qRT-polymerase chain reaction showed that the ZtPPO1 transcripts were highly expressed during larval-prepupal transition and in the haemolymph. When the larvae were fed a 1.66% kojic acid (KA)-containing diet, the levels of the ZtPPO1 transcripts significantly increased by 2.79- and 3.39-fold in the whole larvae and cuticles, respectively, while the corresponding PO activity was significantly reduced; in addition, the larval and pupal durations were significantly prolonged; pupal weights were lowered; and abnormal phenotypes were observed. An in vitro inhibition experiment indicated that KA was an effective competitive inhibitor of PO in Z. tau. Additionally, the functional analysis showed that 20E could significantly up-regulate the expression of ZtPPO1, induce lower pupal weight, and advance pupation. Knockdown of the ZtPPO1 gene by RNAi significantly decreased mRNA levels after 24 h and led to low pupation rates and incomplete pupae with abnormal phenotypes during the larval-pupal interim period. These results proved that PO is important for the normal growth of Z. tau and that KA can disrupt the development of this pest insect.

Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors.

Matriptase is a member of the type-II transmembrane serine protease (TTSP) family and plays a crucial role in the development and maintenance of epithelial tissues. As all chymotrypsin-like serine proteases, matriptase is synthesized as a zymogen (proform), requiring a cleavage event for full activity. Recent studies suggest that the zymogen of matriptase possesses enough catalytic activity to not only facilitate autoactivation, but also carry out its in vivo functions, which include activating several proteolytic and signaling cascades. Inhibition of zymogen matriptase may therefore be a highly effective approach for limiting matriptase activity. To this end, here we sought to characterize the catalytic activity of human zymogen matriptase and to develop mAb inhibitors against this enzyme form. Using a mutated variant of matriptase in which the serine protease domain is locked in the zymogen conformation, we confirmed that the zymogen form of human matriptase has catalytic activity. Moreover, the crystal structure of the catalytic domain of zymogen matriptase was solved to 2.5 Å resolution to characterize specific antibody-based matriptase inhibitors and to further structure-based studies. Finally, we describe the first antibody-based competitive inhibitors that target both the zymogen and activated forms of matriptase. We propose that these antibodies provide a more efficient way to regulate matriptase activity by targeting the protease both before and after its activation and may be of value for both research and preclinical applications.

Biophysical evidence for differential gallated green tea catechins binding to membrane type-1 matrix metalloproteinase and its interactors.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP which triggers intracellular signaling and regulates extracellular matrix proteolysis, two functions that are critical for tumor-associated angiogenesis and inflammation. While green tea catechins, particularly epigallocatechin gallate (EGCG), are considered very effective in preventing MT1-MMP-mediated functions, lack of structure-function studies and evidence regarding their direct interaction with MT1-MMP-mediated biological activities remain. Here, we assessed the impact in both cellular and biophysical assays of four ungallated catechins along with their gallated counterparts on MT1-MMP-mediated functions and molecular binding partners. Concanavalin-A (ConA) was used to trigger MT1-MMP-mediated proMMP-2 activation, expression of MT1-MMP and of endoplasmic reticulum stress biomarker GRP78 in U87 glioblastoma cells. We found that ConA-mediated MT1-MMP induction was inhibited by EGCG and catechin gallate (CG), that GRP78 induction was inhibited by EGCG, CG, and gallocatechin gallate (GCG), whereas proMMP-2 activation was inhibited by EGCG and GCG. Surface plasmon resonance was used to assess direct interaction between catechins and MT1-MMP interactors. We found that gallated catechins interacted better than their ungallated analogs with MT1-MMP as well as with MT1-MMP binding partners MMP-2, TIMP-2, MTCBP-1 and LRP1-clusterIV. Overall, current structure-function evidence supports a role for the galloyl moiety in both direct and indirect interactions of green tea catechins with MT1-MMP-mediated oncogenic processes.

Targeting the Hemopexin-like Domain of Latent Matrix Metalloproteinase-9 (proMMP-9) with a Small Molecule Inhibitor Prevents the Formation of Focal Adhesion Junctions.

A lack of target specificity has greatly hindered the success of inhibitor development against matrix metalloproteinases (MMPs) for the treatment of various cancers. The MMP catalytic domains are highly conserved, whereas the hemopexin-like domains of MMPs are unique to each family member. The hemopexin-like domain of MMP-9 enhances cancer cell migration through self-interaction and heterointeractions with cell surface proteins including CD44 and α4ß1 integrin. These interactions activate EGFR-MAP kinase dependent signaling that leads to cell migration. In this work, we generated a library of compounds, based on hit molecule N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, that target the hemopexin-like domain of MMP-9. We identify N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-ylthio)butanamide, 3c, as a potent lead (Kd = 320 nM) that is specific for binding to the proMMP-9 hemopexin-like domain. We demonstrate that 3c disruption of MMP-9 homodimerization prevents association of proMMP-9 with both α4ß1 integrin and CD44 and results in the dissociation of EGFR. This disruption results in decreased phosphorylation of Src and its downstream target proteins focal adhesion kinase (FAK) and paxillin (PAX), which are implicated in promoting tumor cell growth, migration, and invasion. Using a chicken chorioallantoic membrane in vivo assay, we demonstrate that 500 nM 3c blocks cancer cell invasion of the basement membrane and reduces angiogenesis. In conclusion, we present a mechanism of action for 3c whereby targeting the hemopexin domain results in decreased cancer cell migration through simultaneous disruption of α4ß1 integrin and EGFR signaling pathways, thereby preventing signaling bypass. Targeting through the hemopexin-like domain is a powerful approach to antimetastatic drug development.

Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation.

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.

Inhibition of pro-/active MMP-2 by green tea catechins and prediction of their interaction by molecular docking studies.

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Structural basis for the Zn2+ inhibition of the zymogen-like kallikrein-related peptidase 10.

Although kallikrein-related peptidase 10 (KLK10) is expressed in a variety of human tissues and body fluids, knowledge of its physiological functions is fragmentary. Similarly, the pathophysiology of KLK10 in cancer is not well understood. In some cancer types, a role as tumor suppressor has been suggested, while in others elevated expression is associated with poor patient prognosis. Active human KLK10 exhibits a unique, three residue longer N-terminus with respect to other serine proteases and an extended 99-loop nearly as long as in tissue kallikrein KLK1. Crystal structures of recombinant ligand-free KLK10 and a Zn2+ bound form explain to some extent the mixed trypsin- and chymotrypsin-like substrate specificity. Zn2+-inhibition of KLK10 appears to be based on a unique mechanism, which involves direct binding and blocking of the catalytic triad. Since the disordered N-terminus and several loops adopt a zymogen-like conformation, the active protease conformation is very likely induced by interaction with the substrate, in particular at the S1 subsite and at the unusual Ser193 as part of the oxyanion hole. The KLK10 structures indicate that the N-terminus, the nearby 75-, 148-, and the 99-loops are connected in an allosteric network, which is present in other trypsin-like serine proteases with several variations.

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors.

The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

Lvserpin3 is involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

Serine protease inhibitor, represented by serpin, plays an important inhibitory role on proteases involved in the immune responses. To clarify the immune characterizations of serpin, a novel serpin (Lvserpin3) encoding for 410 amino acids with a 23-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Lvserpin3 expressed strongest in hepatopancreas, and was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Suppression of Lvserpin3 by dsRNA led to a significant increase in the transcripts of LvPPAF, LvproPO and phenoloxidase (PO) activity, and also led to the high cumulative mortality. The recombinant Lvserpin3 protein (rLvserpin3) inhibited the proteases secreted by M. lysodeikticus and Bacillus subtilis, and further exhibited inhibitory role on the growth of B. subtilis and M. lysodeikticu. Moreover, rLvserpin3 was found to be able to block the activation of prophenoloxidase system. Taken together, the results imply that Lvserpin3 may be involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

Protective properties of ginsenoside Rb3 against UV-B radiation-induced oxidative stress in HaCaT keratinocytes.

This work aimed to evaluate the skin anti-photoaging properties of ginsenoside Rb3 (Rb3), one of the main protopanaxdiol-type ginsenosides from ginseng, in HaCaT keratinocytes. The skin anti-photoaging activity was assessed by analyzing the levels of reactive oxygen species (ROS), pro-matrix metalloproteinase-2 (proMMP-2), pro-matrix metalloproteinase-9 (proMMP-9), total glutathione (GSH), and superoxide dismutase (SOD) activity as well as cell viability in HaCaT keratinocytes under UV-B irradiation. When HaCaT keratinocytes were exposed to Rb3 prior to UV-B irradiation, Rb3 exhibited suppressive activities on UV-B-induced ROS, proMMP-2, and proMMP-9 enhancements. On the contrary, Rb3 displayed enhancing activities on UV-B-reduced total GSH and SOD activity levels. Rb3 could not interfere with cell viabilities in UV-B-irradiated HaCaT keratinocytes. Rb3 plays a protective role against UV-B-induced oxidative stress in human HaCaT keratinocytes, proposing its potential skin anti-photoaging properties.

The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab, Callinectes sapidus.

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.

Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease.

Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae.

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active.

Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 µM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.

Exploration of protein-protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies.

In Prophenoloxidase (ProPO) cascade, two targets namely serine protease and α-2-macroglobulin are key regulators involved in the defense system of crustaceans. In biological systems, routine role of cell systems requires the understanding in protein-protein interactions through experimental and theoretical concepts, which might yield useful insights into the cellular responses. Response of cells to regulating the immune system is governed by the interactions-involved biomolecular simulations. Unfortunately, studies on the inhibitors (SP and α-2M) that negatively regulate the proPO system or melanization in penaeid shrimp are not yet available. In order to understand how these interactions change the proPO mechanism in Indian white shrimp Fenneropenaeus indicus was determined. In F. indicus, innate immune system is in a sensitive balance of intricate interactions; elucidating these interactions by the integration of in silico and in vitro has great potential. We have determined the expression of both the SP and α-2M enzymes in regulatory mechanism, which are analyzed through qRT-PCR, protein-protein docking, and simulation studies. From this work, we propose a novel approach for studying an organism at the systems level by integrating genome-wide computational analysis and the gene expression data.

Sodium fluoride inhibits MMP-2 and MMP-9.

The importance of fluoride (F) in preventing dental caries by favorably interfering in the demineralization-remineralization processes is well-established, but its ability to inhibit matrix metalloproteinases (MMPs), which could also help to prevent dentin caries, has not been investigated. This study assessed the ability of F to inhibit salivary and purified human gelatinases MMPs-2 and -9. Saliva was collected from 10 healthy individuals. Pooled saliva was centrifuged, and supernatants were incubated for 1 hr at 37°C and subjected to zymography. Sodium fluoride (50-275 ppm F) was added to the incubation buffer. The reversibility of the inhibition of MMPs-2 and -9 by NaF was tested by the addition of NaF (250-5,000 ppm F) to the incubation buffer, after which an additional incubation was performed in the absence of F. F decreased the activities of pro- and active forms of salivary and purified human MMPs in a dose-response manner. Purified gelatinases were completely inhibited by 200 ppm F (IC50 = 100 and 75 ppm F for MMPs-2 and -9, respectively), and salivary MMP-9 by 275 ppm F (IC50 = 200 ppm F). Inhibition was partially reversible at 250-1,500 ppm F, but was irreversible at 5,000 ppm F. This is the first study to describe the ability of NaF to inhibit MMPs completely.

Vitamin D3 inhibits expression and activities of matrix metalloproteinase-2 and -9 in human uterine fibroid cells.

STUDY QUESTION: Can biologically active vitamin D3 [1,25(OH)2D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells? SUMMARY ANSWER: 1,25(OH)2D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells. WHAT IS KNOWN ALREADY: Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)2D3 can regulate the expression and activities of MMPs in human uterine fibroid cells. STUDY DESIGN, SIZE, DURATION: Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)2D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay. MAIN RESULTS AND THE ROLE OF CHANCE: 1-1000 nM 1,25(OH)2D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)2D3. 1,25(OH)2D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)2D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)2D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)2D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable. LIMITATIONS, REASONS FOR CAUTION: This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals an important biological function of 1,25(OH)2D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)2D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.