Inhibition Of Proteolysis Research Articles

My Times with Professor Igor Kregar and some Hydrolytic Enzymes.

The late Professor Igor Kregar and this author had several overlapping biochemical research interests. One focus was the glycoside hydrolase, lysozyme, and the other was proteolytic enzymes and their inhibitors, in particular those present in tissues from insects and plants. Regarding lysozyme our results helped to understand its catalytic mechanism and the carboxylic acid functional groups involved. Another area was insect-plant interactions involving defensive responses of plants to insect feeding via proteolytic enzyme inhibitors. Those results can be utilized in transgenic plant and seed biotechnological applications, which would help to reduce damage to plants and seeds caused by coleopteran and other insect pests. Also described are some cultural and travel interactions that the author benefited from his friendship with Professor Kregar.

Nuclear softening is essential for protease-independent migration

During amoeboidal migration, cancer cells migrate in a protease-independent manner by squeezing through pre-existing gaps in the extracellular matrix (ECM). However, the extent to which cells alter their physical properties in order to sustain this mode of migration remains unclear. Here, we address this question by documenting biophysical changes in the properties of highly invasive MDA-MB-231 and HT-1080 cells upon inhibition of pericellular proteolysis. Remarkably, treatment with the broad spectrum MMP inhibitor GM6001 not only induces cell rounding and loss of actomyosin contractility, but also induces nuclear softening via increased phosphorylation of the nuclear membrane protein lamin A/C. Though nuclear softening is necessary for sustaining migration through sub-nuclear sized transwell pores, it is not sufficient. In addition, baseline levels of contractility mediating pore entry and peri-nuclear actin inside the pores mediating pore migration are also required. Taken together, our results suggest that protease-independent migration through sub-nuclear sized pre-existing tracks is enabled by deformation of a softened nucleus by contractility and the peri-nuclear actin network.

The Effects of UCH-L3 on the Biological Behaviours of Breast Cancer: Correlation with the Expressions of HIF-1α

Projective: HIF-1α is highly expressed in the triple negative breast cancer cell line (MDA-MB-231), which is lack of the expression of ER,PR and HER2 and exhibits high invasive and metastatic ability. Previous study detected that siRNA targeting HIF-1α in MDA-MB-231 restrained the cell growth and the abilities of immigration and invasion, and promoted apoptosis. UCH-L3 protein was found to be one of the differential proteins detected by Bidirectional gel Electrophoresis and Proteomics in the cells with HIF-1α siRNA cells comparing to no-siRNA cells. The objective of this study is to probe the effect of UCH-L3 on the biological behaviors of the triple negative breast cancer cell line (MDA-MB-231). Methods: Over or blocking expressions of UCH-L3 were established by the transfections with lentiviral constitutive vector and siRNA targeting UCH-L3 respectively. Real time quantitative PCR and Western blot or Co-ip were used to detect the mRNA and proteins. CCK8, and clone formation assays were to evaluate the cell growth and clonality. Matrigel Transwell and Would Scratch assay were used to estimate the cell invasion and mobility. Results: The over-expression of UCH-L3 inhibited the cell growth and clonality, weaken the abilities of cell immigration and invasion, and lowered the expression of free and ubiquitined HIF-1α in MDA-MB-231 cells with lentiviral vector comparing to those in the cells with control vector. Application of proteolytic enzyme inhibitor, MG132 increased the protein level of UCH-L3 but still decreased the protein level of HIF-1α in the cells with UCH-L3 over-expression. Blocking UCH-L3 expression by siRNA technique increased the expression of HIF-1α properly. Conclusion: high expression of UCH-L3 showed an inhibitory effect on the biological behaviors of triple negative breast cancer cell and negative effect on HIF-1α expression, implying that UCH-L3 likely to be a therapeutic strategy for triple negative breast cancer.

Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy

Brain tumor patients commonly present with epileptic seizures. We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain. The reduced firing is due to the degradation of perineuronal nets (PNNs) that surround FSNs. We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies. Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release. These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates. Disruption of PNNs may similarly account for excitation-inhibition imbalances in other forms of epilepsy and PNN protection through proteolytic inhibition may provide therapeutic benefits.

СТАН СИСТЕМИ ПРОТЕЇНАЗИ/ІНГІБІТОРИ ПРОТЕЇНАЗ У ЩУРІВ У ДИНАМІЦІ ІММОБІЛІЗАЦІЙНОГО СТРЕСУ НА ТЛІ ГІПОТИРЕОЗУ

Вступ. У патогенезі багатьох патологічних станів важливе місце посідають порушення протеїназо-інгібіторної системи. При відсутності належного контролю за протеолізом розвиваються патологічні стани, що супроводжуються виникненням деструктивних, запальних та імунних реакцій. Ряд дослідників встановив захисну дію йодовмісних тиреоїдних гормонів при стресі, які реалізуються в результаті їх взаємодії з клітинним геномом, що призводить до стимуляції локальних стрес-лімітуючих систем.
 Мета дослідження – встановити особливості функціонування системи протеїнази/інгібітори протеїназ за умов іммобілізаційного стресу на тлі зниження рівня йодовмісних гормонів щитоподібної залози.
 Методи дослідження. Гіпотиреоз моделювали, щоденно вводячи тваринам per os тиреостатик мерказоліл (“Здоров’я”, Україна) у дозі 25 мг/кг протягом 21-ї доби. Гострий іммобілізаційний стрес моделювали шляхом прив’язування піддослідних щурів у положенні на спині за 4 кінцівки без обмеження рухо­мості голови тривалістю 3 год.Для дослідження концентрації йодовмісних гормонів щитоподібної залози, активності протеїназної системи і вмісту інгібіторів протеолізу використано спектрофотометричні та імуноферментні методи.
 Результати й обговорення. Досліджено вплив іммобілізаційного стресу в щурів з попередньо змодельованим гіпотиреозом на показники протеїназо-інгібіторної системи крові. На стадії тривоги розвитку стрес-реакції в евтиреоїдних тварин встановлено підвищення протеолітичної активності крові на тлі збільшення вмісту α1-інгібітора протеаз та α2-макроглобуліну, що вказує на посилення антипротеолітичного потенціалу для стримування розвитку деструкції.На стадії резистентності відбувалася стабілізація протеолітичної активності крові. Однак при тривалому стресі (стадія виснаження) вона знову достовірно зростала на тлі зниження активності інгібіторів протеаз, що свідчить про виснаження захисного інгібіторного резерву. У тварин з гіпотиреозом на всіх стадіях розвитку стрес-реакції мало місце достовірне підвищення протеолітичної активності крові, причому показники інгібіторів протеаз достовірно зменшувалися. Це призводило до різкого зростання індексу протеолізу, що відбувалося на тлі збільшення проникності лізосомальних мембран.
 Висновок. На тлі гіпотиреозу спостерігають інтенсивніше, ніж у евтиреоїдних тварин, зростання протеолітичної активності крові на фоні пригнічення антипротеазної активності та збільшення проникності лізосомальних мембран.

Proteolysis is the most fundamental property of malignancy and its inhibition may be used therapeutically (Review).

The mortality rates of cancer patients decreased by ~1.5% per year between 2001 and 2015, although the decrease depends on patient sex, ethnic group and type of malignancy. Cancer remains a significant global health problem, requiring a search for novel treatments. The most common property of malignant tumors is their capacity to invade adjacent tissue and to metastasize, and this cancer aggressiveness is contingent on overexpression of proteolytic enzymes. The components of the plasminogen activation system (PAS) and the metal-loproteinase family [mainly matrix metalloproteinases (MMPs)] are overexpressed in malignant tumors, driving the local invasion, metastasis and angiogenesis. This is the case for numerous types of cancer, such as breast, colon, prostate and oral carcinoma, among others. Present chemotherapeutics agents typically attack all dividing cells; however, for future therapeutic agents to be clinically successful, they need to be highly selective for a specific protein(s) and act on the cancerous tissues without adverse systemic effects. Inhibition of proteolysis in cancerous tissue has the ability to attenuate tumor invasion, angiogenesis and migration. For that purpose, inhibiting both PAS and MMPs may be another approach, since the two groups of enzymes are overexpressed in cancer. In the present review, the roles and new findings on PAS and MMP families in cancer formation, growth and possible treatments are discussed.

Optimization of proteolysis and angiotensin converting enzyme inhibition activity in a model cheese using response surface methodology

Adjunct cultures including lactobacilli play important role in cheese proteolysis and bioactivity including Angiotensin Converting Enzyme (ACE)-inhibition and antioxidant activity. Four species of lactobacilli was used in the manufacture of model cheese and these were salted at different levels and the resultant cheeses were ripened at different time and temperature to understand the effect of salting and ripening conditions. The effects of the use of various of lactobacilli (Lactobacillus casei, Lb. helveticus, Lb. delbrueckii ssp. bulgaricus and Lb. plantarum), salt concentrations (1, 2, 3, 4 and 5%), ripening times (1, 30, 60, 90 and 120 days) and temperatures (5, 10, 15 and 20 °C) were investigated. The experiments were designed to determine the optimal salt concentration, ripening times and temperatures for optimal proteolysis, the highest levels of ACE-inhibition and antioxidant activity. The highest level of proteolysis and ACE-inhibiting activity in the model cheese was observed when ripening temperatures were between 8 and 16 °C, salt concentration at 2.5–3.0% and with a ripening period of about 60 days. The use of each lactobacilli contributed to the proteolysis in different manner; for example, ACE-inhibiting activity and antioxidant activity in the model cheese.

Effect of nitric oxide and calpastatin on the inhibition of µ-calpain activity, autolysis and proteolysis of myofibrillar proteins

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating µ-calpain activity in the presence of calpastatin.

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

Pharmacological correction of the hemostasis system for the surgical treatment of bitches with tumours of the mammary gland

We carried out the clinical validation of pathogenetically substantiated protocols for the treatment of female dogs with tumour lesions of the mammary gland. The positive effect of corrective therapy was characterized by a decrease in the physiological level of fibrinogen: in benign tumours in 10 days, malignant – in 14 days against the background of its stable high level in control animals with malignant neoplasia. In the postoperative period in experimental animals, the functioning of the internal coagulation unit was restored, as evidenced by the normalization of the activated partial thromboplastin time for benign tumours by days 10–14, malignant – by 14 days. In control patients, these changes were detected only for benign neoplasms. Shifts of the external mechanism of blood coagulation in the experimental groups were eliminated in benign neoplasia cases in 10 days, malignant cases in 14 days against the background of severe disorders in this link in control patients throughout the observation period. The positive effect of complex treatment regimens is confirmed by the restoration of total fibrinolytic activity by days 10–14 due to the normalization in the same terms of the plasminogen activator and tissue plasminogen activator by days 3–7. In control animals, the total fibrinolytic activity was consistent with those of clinically healthy animals only for benign neoplasms. The balancing of coagulation and fibrinolytic mechanisms was accompanied by a decrease in the activity of proteolytic inhibitors: α2-macroglobulin in experimental groups with benign neoplasia by day 10, malignant – by days 10–14, and control – respectively, by days 3 and 14; α1-proteinase inhibitor in all groups by day 3. Concentration of nitric oxide after extirpation of benign tumors already at day 3 of the postoperative period in all groups corresponded to the indexes of clinically healthy dogs, with the removal of malignant tumours – in experimental animals at day 3, control – at day 14. The content of malondialdehyde and ceruloplasmin against the background of pharmacological correction was restored by days 10–14, whereas in control animals – only with benign neoplasms by day 14. Postoperative pharmacological correction of the hemostasis system can significantly improve the results of treatment in cancer patients.

Proteomic identification and purification of seed proteins from native Amazonian species displaying antifungal activity.

Seeds of native species from the rain forest (Amazon) are source of chitinases and their protein extracts exhibited strong and broad antifungal activity. Numerous plant species native to the Amazon have not yet been chemically studied. Studies of seeds are scarcer, since adversities in accessing study areas and seasonality pose constant hurdles to systematic research. In this study, proteins were extracted from seeds belonging to endemic Amazon species and were investigated for the first time. Proteolytic activity, peptidase inhibitors, and chitinases were identified, but chitinolytic activity predominated. Four proteins were purified through chromatography and identified as lectin and chitinases by MS/MS analyses. The proteins were examined for inhibition of a phytopathogen (Fusarium oxysporum). Analyses by fluorescence microscopy suggested binding of propidium iodide to DNA of fungal spores, revealing that spore integrity was lost when accessed by the proteins. Further structural and functional analyses of defensive proteins belonging to species facing highly complex ecosystems such as Amazonia should be conducted, since these could provide new insights into specificity and synergism involving defense proteins of plants submitted to a very complex ecosystem.

Proteolysis inhibition by hibernating bear serum leads to increased protein content in human muscle cells

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

The role of the peritrophic matrix and red blood cell concentration in Plasmodium vivax infection of Anopheles aquasalis

BackgroundPlasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development.ResultsAbsence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity.ConclusionsThis study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection.

Macrocyclic θ-defensins suppress tumor necrosis factor-α (TNF-α) shedding by inhibition of TNF-α–converting enzyme

Theta-defensins (θ-defensins) are macrocyclic peptides expressed exclusively in granulocytes and selected epithelia of Old World monkeys. They contribute to anti-pathogen host defense responses by directly killing a diverse range of microbes. Of note, θ-defensins also modulate microbe-induced inflammation by affecting the production of soluble tumor necrosis factor (sTNF) and other proinflammatory cytokines. Here, we report that natural rhesus macaque θ-defensin (RTD) isoforms regulate sTNF cellular release by inhibiting TNF-α-converting enzyme (TACE; also known as adisintegrin and metalloprotease 17; ADAM17), the primary pro-TNF sheddase. Dose-dependent inhibition of cellular TACE activity by RTDs occurred when leukocytes were stimulated with live Escherichia coli cells as well as numerous Toll-like receptor agonists. Moreover, the relative inhibitory potencies of the RTD isoforms strongly correlated with their suppression of TNF release by stimulated blood leukocytes and THP-1 monocytes. RTD isoforms also inhibited ADAM10, a sheddase closely related to TACE. TACE inhibition was abrogated by introducing a single opening in the RTD-1 backbone, demonstrating that the intact macrocycle is required for enzyme inhibition. Enzymologic analyses showed that RTD-1 is a fast binding, reversible, non-competitive inhibitor of TACE. We conclude that θ-defensin-mediated inhibition of pro-TNF proteolysis by TACE represents a rapid mechanism for the regulation of sTNF and TNF-dependent inflammatory pathways. Molecules with structural and functional features mimicking those of θ-defensins may have clinical utility as TACE inhibitors for managing TNF-driven diseases.

How proteolytic inhibitors interact with dentin on glass-fiber post luting over 6 months

Enzyme inhibitors minimize the degradation of unprotected collagen of dentin promoted by matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs). As the evidence of their effect on the root canal is limited, this study aimed to evaluate the role of EDTA, chlorhexidine and E-64 as antiproteolytic agents on the bond strength (BS) of glass-fiber posts in root canals. Ninety-six bovine roots were distributed in groups for each time point (n = 8). Adper Scotchbond Multipurpose (MP)/ RelyX ARC system was used to lute the post according to the treatment: negative control (NC)- water, EDTA- 17% ethylenediaminetetraacetic acid, CHX- 2% digluconate chlorhexidine, E-64-5- 5μM E-64, E-64-10- 10μM E-64 and positive control (PC)- MP associated with activator/ catalyst. Then, slices were subjected to push-out test (0.5mm/min) after 24h/6 mons. Data were analyzed by three-way ANOVA/Tukey tests. Failure modes were analyzed (40×). The factors treatment, time, root canal third and the interaction between treatment and time were statistically significant. At 24h, no negative interactions were observed among the root dentin, bonding system and post. At 6 mons, CHX improved the BS for middle and apical root thirds. CHX was able to promote beneficial BS after 6 mons, which was not noted for any other tested enzyme inhibitors.

L-arginine ingestion inhibits eccentric contraction-induced proteolysis and force deficit via S-nitrosylation of calpain.

It has been shown that calpains are involved in the proteolysis of muscle proteins that occurs with eccentric contraction (ECC) and that exogenously applied nitric oxide decreases the calpain‐mediated proteolysis. The aim of this study was to examine the effects of ingestion of l‐arginine (ARG), a nitric oxide precursor, on ECC‐related calpain activation. In the first and second experiments, male Wistar rats were given ARG in water for 7 days starting from 3 days before the ECC protocol (average ingestion, ~600 mg kg‐body wt−1 day−1). Tibialis anterior muscles underwent 200 repeated ECCs and, subsequently, were excised 3 days later. Whole muscle analyses (the first experiment) revealed that ARG attenuated ECC‐induced force deficit and autolysis of calpain‐1, and increased the amounts of S‐nitrosylated calpain‐1. Regarding ryanodine receptor (RyR) and dihydropyridine receptor (DHPR), ECC‐induced proteolysis was completely inhibited by ARG, whereas the inhibition was partial for junctophilin‐1 (JP1). Skinned fiber analyses (the second experiment) showed that ARG also inhibited ECC‐elicited reductions in the ratio of depolarization‐induced to maximum Ca2+‐activated force. In the third experiment, homogenates of rested muscles were treated with S‐nitrosylating agent, S‐nitrosoglutathione (GSNO), and/or high Ca2+ concentration ([Ca2+]). Treatment with high [Ca2+] and without GSNO produced proteolysis of RyR, DHPR, and JP1. On the other hand, treatment with high [Ca2+] and GSNO caused complete inhibition of RyR and DHPR proteolysis and partial inhibition of JP1 proteolysis. These results indicate that ARG ingestion can attenuate ECC‐induced proteolysis of Ca2+ regulatory proteins and force deficit by decreasing calpain activation via S‐nitrosylation.

Comparative transcriptome analysis provides insights of anti-insect molecular mechanism of Cassia obtusifolia trypsin inhibitor against Pieris rapae.

Pieris rapae, a serious Lepidoptera pest of cultivated crucifers, utilizes midgut enzymes to digest food and detoxify secondary metabolites from host plants. A recombinant trypsin inhibitor (COTI) from nonhost plant, Cassia obtusifolia, significantly decreased activities of trypsin-like proteases in the larval midgut on Pieris rapae and could suppress the growth of larvae. In order to know how COTI took effect, transcriptional profiles of P. rapae midgut in response to COTI was studied. A total of 51,544 unigenes were generated and 45.86% of which had homologs in public databases. Most of the regulated genes associated with digestion, detoxification, homeostasis, and resistance were downregulated after ingestion of COTI. Meanwhile, several unigenes in the integrin signaling pathway might be involved in response to COTI. Furthermore, using comparative transcriptome analysis, we detected differently expressing genes and identified a new reference gene, UPF3, by qRT-polymerase chain reaction (PCR). Therefore, it was suggested that not only proteolysis inhibition, but also suppression of expression of genes involved in metabolism, development, signaling, and defense might account for the anti-insect resistance of COTI.

Green Tea Polyphenols Decrease Strecker Aldehydes and Bind to Proteins in Lactose-Hydrolyzed UHT Milk.

The effect of epigallocatechin gallate enriched green tea extract (GTE) on flavor, Maillard reactions and protein modifications in lactose-hydrolyzed (LH) ultrahigh temperature (UHT) processed milk was examined during storage at 40 °C for up to 42 days. Addition of GTE inhibited the formation of Strecker aldehydes by up to 95% compared to control milk, and the effect was similar when GTE was added either before or after UHT treatment. Release of free amino acids, caused by proteolysis, during storage was also decreased in GTE-added milk either before or after UHT treatment compared to control milk. Binding of polyphenols to milk proteins was observed in both fresh and stored milk samples. The inhibition of Strecker aldehyde formation by GTE may be explained by two different mechanisms; inhibition of proteolysis during storage by GTE or binding of amino acids and proteins to the GTE polyphenols.

The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database.

The MEROPS database (http://www.ebi.ac.uk/merops/) is an integrated source of information about peptidases, their substrates and inhibitors. The hierarchical classification is: protein-species, family, clan, with an identifier at each level. The MEROPS website moved to the EMBL-EBI in 2017, requiring refactoring of the code-base and services provided. The interface to sequence searching has changed and the MEROPS protein sequence libraries can be searched at the EMBL-EBI with HMMER, FastA and BLASTP. Cross-references have been established between MEROPS and the PANTHER database at both the family and protein-species level, which will help to improve curation and coverage between the resources. Because of the increasing size of the MEROPS sequence collection, in future only sequences of characterized proteins, and from completely sequenced genomes of organisms of evolutionary, medical or commercial significance will be added. As an example, peptidase homologues in four proteomes from the Asgard superphylum of Archaea have been identified and compared to other archaean, bacterial and eukaryote proteomes. This has given insights into the origins and evolution of peptidase families, including an expansion in the number of proteasome components in Asgard archaeotes and as organisms increase in complexity. Novel structures for proteasome complexes in archaea are postulated.

Role of Proteolytic Enzyme Inhibitors on Carious and Eroded Dentin Associated With a Universal Bonding System.

The aim of this study was to evaluate the effect of proteolytic inhibitors on the bond strength of a universal adhesive system (etch-and-rinse mode) applied to artificial carious and eroded dentin. Ninety molars were prepared and randomly divided into three groups according to the substrate: N, no challenges; ACD, artificial carious dentin simulation and ERO, artificial erosion simulation with orange juice. All groups were redivided into three subgroups according to the dentin pretreatment: W, water; CHX, 2% digluconate chlorhexidine; and E-64 (trans-epoxysuccinyl-L-leucylamido-[4-guanidino] butane), 5 μM E-64 inhibitor. They constituted a total of nine groups (n=10): N-W, N-CHX, N-E64, ACD-W, ACD-CHX, ACD-E64, ERO-W, ERO-CHX, and ERO-E64. All specimens were restored with Adper Single Bond Universal/Filtek Z250. Beams (0.64 mm2) were obtained and subjected to the microtensile test (μTBS) in a universal testing machine at 0.5 mm/min. The failure mode of the interfaces was determined by optical microscopy (40× magnification). Data were statistically analyzed by three-way analysis of variance and Tukey tests (p<0.05). All individual factors (p<0.0001) and the interaction between substrate and treatment (p=0.0011) and between substrate and time (p=0.0003) were statistically significant. The caries substrate contributed negatively to bond strength. Chlorhexidine reduced bond strength for normal and eroded conditions. Only the normal substrate was negatively affected by time despite the pretreatment. The universal bonding system appears to be a promising bonding strategy for the maintenance of bond strength to affected dentin. E-64 did not affect bonding to the dentin in contrast to the use of chlorhexidine, which, when associated with the universal system, did affect the microtensile bond strength for artificial carious dentin.