Proprotein convertase subtilisin/kexin 9 inhibitor downregulates microRNA-130a-3p expression in hepatocytes to alleviates atherosclerosis progression.

Proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors have been shown to regulate lipid metabolism and reduce the risk of cardiovascular events. This study explores the effect and potential mechanism of PCSK9 inhibitors on lipid metabolism and coronary atherosclerosis. HepG2 cells were incubated with PCSK9 inhibitor. ApoE-/- mice were fed with a high fat to construct an atherosclerosis model, and then treated with PCSK9 inhibitor (8 mg/kg for 8 w). PCSK9 inhibitor downregulated microRNA (miRNA)-130a-3p expression in a dose-dependent manner. And, miR-130a-3p could bind directly to the 3' untranslated region (3'-UTR) region of LDLR to down-regulate LDLR expression in HepG2 cells, as confirmed by the luciferase reporter gene assay. In addition, miR-130a-3p overexpression significantly attenuated the promoting effect of PCSK9 inhibitor on LDLR and DiI-LDL uptake in HepG2 cells. More importantly, in vivo experiments confirmed that PCSK9 inhibitor could significantly inhibit miR-130a-3p levels and promote LDLR expression in liver tissues, thus regulating serum lipid profile and alleviating the progression of coronary atherosclerosis. PCSK9 inhibitor could moderately improve coronary atherosclerosis by regulating miR-130a-3p/LDLR axis, providing an exploitable strategy for the treatment of coronary atherosclerosis.

Protective Mechanism of Proprotein Convertase Subtilisin-Like Kexin Type 9 Inhibitor on Rats with Middle Cerebral Artery Occlusion-Induced Cerebral Ischemic Infarction.

The aim of this study was to research the mechanism of proprotein convertase subtilisin-like kexin type 9 (PCSK9) inhibitor in neural protective effect on rat cerebral ischemic reperfusion injury (I/RI). The transient middle cerebral artery occlusion (tMCAO) model of rats was prepared by the suture method, and PCSK9 inhibitor was injected intraperitoneally immediately after I/R. The rats were scored for neurological deficits and the cerebral infarction volume was measured. The brain tissues were collected and western blot (WB) was used to detect the expression of PCSK9. The rat cortical neural stem cells were treated with oxygen glucose deprivation (OGD) to establish a cell model of ischemia/reperfusion. WB was used to detect the expression of PCSK9 and the apoptosis-related pathway proteins. After interfering with the expression of PCSK9 siRNA, the cell viability (cell counting kit-8 assay) and apoptosis (TUNEL staining, Annexin V/PI method) were detected, and the cell proliferation was detected by EdU staining and flow cytometry. The expression of PCSK9 in the brain tissue of the MCAO group was dramatically increased. PCSK9 inhibitor can improve neurobehavioral scores and reduce apoptosis and infarct volume. An OGD model of neural stem cells in vitro was constructed. Inhibiting PCSK9 with si-PCSK9 can increase cell viability, promote cell proliferation, and also reduce cell apoptosis. Inhibition of PCSK9 can decrease the cerebral infarct volume in rats with cerebral I/RI and improve the neural function. Mechanically, inhibition of PCSK9 can lead to the decrease of nerve cell apoptosis and promotion of cell proliferation.

Metformin enhances LDL-cholesterol uptake by suppressing the expression of the pro-protein convertase subtilisin/kexin type 9 (PCSK9) in liver cells.

PURPOSE: Metformin (MF) intake associates with reduced levels of circulating low-density lipoprotein-cholesterol (LDL-C). This has been attributed to the activation of AMPK, which differentially regulates the expression of multiple genes involved in cholesterol synthesis and trafficking. However, the exact mechanism underlying the LDL-C lowering effect of MF remains ambiguous. METHODS: MF-treated Hep-G2 and HuH7 cells were evaluated for cell viability and the expression status of key lipid metabolism-related genes along with LDL-C uptake efficiency. RESULTS: MF treatment resulted in decreased expression and secretion of PCSK9, increased expression of LDLR and enhanced LDL-C uptake in hepatocytes. It also resulted in increased expression of activated AMPK (p-AMPK) and decreased expression of SREBP2 and HNF-1α proteins. Transcriptomic analysis of MF-treated Hep-G2 cells confirmed these findings and showed that other key lipid metabolism-related genes including those that encode apolipoproteins (APOB, APOC2, APOC3 and APOE), MTTP and LIPC are downregulated. Lastly, MF treatment associated with reduced HMG-CoA reductase expression and activity. CONCLUSIONS: These findings suggest that MF treatment reduces circulating LDL-C levels by suppressing PCSK9 expression and enhancing LDLR expression; hence the potential therapeutic utility of MF in hypercholesterolemia.

Evaluation of the effects of natural isoquinoline alkaloids on low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) in hepatocytes, as new potential hypocholesterolemic agents.

Nine different isoquinoline alkaloids, berberine, govaniadine, stylopine, adlumine, adlumidine, bicuculline, sanguinarine, protopine and californidine have been evaluated for their effects on a cellular model of hepatocyte for their effect on low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression compared to simvastatin. Berberine, californidine and govaniadine induced LDLR with an effect similar to 2.5 µM simvastatin. Californidine and berberine at tested doses reduced the expression of PCSK9, with an opposite behaviour to simvastatin on this target. Govaniadine, on the other hand, showed a statin-like effect, although less potently, by increasing both LDLR and PCSK9 levels. Berberine californidine and govaniadine were then tested on the same cellular model to assess possible effect of reduction of total cholesterol, compared to simvastatin. All compounds were able to reduce total cholesterol level in the hepatocytes.

Identification of Multi-Potent Protein Subtilisin A from halophilic bacterium Bacillus firmus VE2.

Screening of halophiles with antimicrobial activity in saltpan soil samples from Nagapattinam district, Tamil Nadu, revealed isolate VE-2 as the most potent, identified as Bacillus firmus strain VE-2 through 16s rRNA gene sequencing. It had an optimum growth condition (OD 3.1) and antimicrobial protein (AMP) production (450 µg/mL) at 37 °C, pH 8, 25% NaCl, and 36 h incubation. SDS-PAGE analysis of the purified AMP showed the molecular weight of 36 kDa. HPLC analysis of the purified AMP showed different amino acids, such as asparagines, alanine, lysine, proline, threonine, glycine, cysteine, serine, aspartic acid leucine, and valine. Further characterization and identification using FT-IR, 2D-PAGE, MALDI-TOF, and in-silico analysis showed that the isolated AMP had the highest similarity to Subtilisin-A. It showed antibacterial activity against clinical bacterial pathogens like S. aureus, S. pyogenes, C. diphtheria, E. coli, and P. aeruginosa with the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration of 2.5 µg/mL and 20 µg/mL and also against various fungal pathogens such as A. niger, A. flavus, C. albicans, C. tropicalis and C. parapsilosis with the MIC and minimum fungicidal concentrations of 1.25-80 µg/mL. The purified AMP had excellent antioxidant potential, showed a scavenging effect against DPPH and Nitric oxide radicals, and displayed anticancer activity against HeLa cell lines with the IC50 values 53 µg/mL. Hence, the purified bioactive antimicrobial peptides (AMP) could also be used in anticancer therapies.

Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes.

We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

The fungal elicitor AsES requires a functional ethylene pathway to activate the innate immunity in strawberry.

Acremonium strictum Elicitor Subtilisin (AsES) is a fungal elicitor that activates innate immunity, conferring disease resistance in strawberry (Fragaria × ananassa Duch.), Arabidopsis and other plant species. The aim of the present work was to evaluate the involvement of the ethylene (ET) signalling pathway in AsES-mediated immune response in strawberry. Ethylene production and expression of the genes responsible for ET synthesis, perception and response were measured after AsES treatment. ROS (H2 O2 ) accumulation and immunity induced by AsES were studied after ET perception was blocked by 1-methylcyclopropene (1-MCP). Biochemical and molecular results showed that AsES induced a marked increase in local and systemic biosynthesis of ET, both in a biphasic manner. Blocking of ET perception by 1-MCP prior to AsES induction reduced production of ROS (H2 O2 ) and prevented AsES from eliciting defence against fungal pathogens having different lifestyles, such as Botrytis cinerea (necrotrophic) and Colletotrichum acutatum (hemibiotrophic). These findings contribute to elucidate the mode of action of the novel elicitor subtilase, AsES, specifically regarding the role of ET signalling in the activation of plant innate immunity, in addition to the multitude of processes regulated by ET in plants.

Evaluation of methods for DNA extraction from Clostridium tyrobutyricum spores and its detection by qPCR.

Clostridium tyrobutyricum is the major agent that causes the blowing defect in cheese due to the germination of its dormant spores during the ripening stage. As a result, many of the affected cheeses show cavities and cracks, which cause the product loss in most cases. Nowadays, there is not a fast method capable of detecting milk contaminated with C. tyrobutyricum spores. The aim of this study has been to develop a fast and reliable method based on real time PCR (qPCR) to detect C. tyrobutyricum spores in raw milk. One of the main limitations has been to find a good procedure for the spore disruption to extract the DNA due to its high resistance. For this reason, different disruption methods have been tested, including chemical agents, bead beating, enzymatic and microwave treatment. Furthermore, an enzymatic treatment with subtilisin was applied for milk clarification and recovery of spores. The comparison of the assayed methods has been made using sterile milk spiked with C. tyrobutyricum spores, obtained in solid or liquid medium. The results showed that microwave treatment followed by a standard DNA purification step was found to be the best disruption method. The Ct values obtained for spores were higher than those found for vegetative cells by qPCR, for the same quantity of DNA. This difference could be due to the action of the Small Acid Soluble Proteins (SASP) in the DNA packaging of spores. Moreover, spores obtained in agar plate were found more resistant to disruption than those obtained in liquid medium. Subtilisin and microwave treatments were found to be successful for DNA extraction from C. tyrobutyricum spores in milk and subsequent identification by qPCR. However, the differences observed between the amplification of DNA from spores obtained in different media and from vegetative cells have to be taken into account to optimize a method for C. tyrobutyricum detection.

A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase).

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans-formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

Structure-Based Prototype Peptides Targeting the Pseudomonas aeruginosa Type VI Secretion System Effector as a Novel Antibacterial Strategy.

The type VI secretion system (T6SS) secretes numerous toxins for bacteria-bacteria competition. TplE is a newly identified trans-kingdom toxin secreted by the T6SS in Pseudomonas aeruginosa, while TplEi neutralizes the toxic effect of TplE to protect bacteria autointoxication. Blocking the interaction of TplE-TplEi could unleash the toxin, causing bacterial cell death. In this study, we applied a crystallographic approach to design a structural-based antimicrobial peptides targeting the interaction of TplE and TplEi. We found that a peptide (designed as "L" peptide based on its shape) derived from TplE can form a crystal complex with TplEi after subtilisin treatment and the crystal structure was solved at 2.2Å. The "L" peptide displays strong binding affinity to TplEi in vitro and can release the TplE toxin to induce bacteria death in vivo. Our findings suggest that as a toxin activator, the "L" peptide could be a possible drug lead for treating P. aeruginosa infection. Our findings provide an example that the T6SS effector and immunity protein could be a potential drug target against bacteria infection.

Effect of exogenous xylanase, amylase, and protease as single or combined activities on nutrient digestibility and growth performance of broilers fed corn/soy diets.

Two trials (a 42-d performance and a 21-d cohort digestibility) were conducted to evaluate the performance and nutrient digestibility of broilers fed corn diets supplemented with exogenous xylanase, amylase, and protease as single or combined activities. A nutritionally adequate, positive control (PC) diet was formulated. The negative control (NC) diet was formulated to be lower in metabolizable energy (∼86 kcal/kg diet) and digestible amino acids (1 to 2%) compared to PC. The other 4 treatments were based on the NC and they were either supplemented with xylanase (X), amylase (A), protease (P), or a combination of X, A, and P (XAP; to provide 2,000 U of X, 200 U of A, and 4,000 U of P/kg diet). All diets were marginal in AvP and Ca and contained a background of phytase (1,000 FTU/kg). In each trial, male broiler (Ross 308) chicks were allocated to the 5 treatments (10 replicates of 20 birds/pen and 9 replicates of 8 birds/cage for the performance and digestibility trials, respectively). In the digestibility trial, ileal digesta was collected on d21 for the determination of nutrient utilization. Data were subjected to one-way ANOVA and means were separated by Tukey's HSD test. Only the XAP improved (P < 0.05) AMEn compared to NC. X, A or XAP improved (P < 0.05) N digestibility and apparent ileal digestible energy (AIDE). Both P and XAP improved N retention. The relative improvement in energy digestibility due to enzyme supplementation was greater at the ileal level than that measured in the excreta. The measured changes on AIDE due to supplemental enzymes were much higher than the sum of calculated contributions from starch, fat, and protein. Supplementation of all enzymes reduced (P < 0.05) ileal flow of soluble rhamnose and mannose relative to NC. In the performance trial, both X and XAP improved (P < 0.05) weight gain (WG) and only XAP improved (P < 0.05) FCR compared to NC during the starter phase (1-21d). Over the entire period (1-42d), WG and FI were not influenced (P > 0.05) by dietary treatments. Both X and XAP had lower (P < 0.05) FCR compared to NC (1.540 and 1.509 vs 1.567, respectively). However, birds fed diet supplemented with XAP had an improved (P < 0.05) FCR compared to birds fed single activities and had similar (P > 0.05) FCR compared to PC. In conclusion, these results suggest a synergistic effect between X, A and P on broiler performance and nutrient digestibility. In the current study, AIDE measurements appeared to overestimate the enzyme response. Calculation of the energy contribution by supplemental enzymes using the improvements in the digestibility of the undigested fraction of starch, fat and protein may be a more accurate measurement for the enzyme response than the absolute response in AIDE.

Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation.

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analyzed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis - dynasore and tat-gluR2(3Y) - were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process.

Effects of exogenous proteases without or with carbohydrases on nutrient digestibility and disappearance of non-starch polysaccharides in broiler chickens.

The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on digestibility and retention of energy and protein, as well as the solubilization and disappearance of non-starch polysaccharides (NSP) from corn-soybean meal based diets fed to broiler chickens. Two hundred eighty-eight Ross 308 male broiler chickens were used for the experiment. On d 14, the birds were weighed and allocated to 6 treatments and 8 replicates per treatment with 6 birds per replicate. Treatments were: 1) corn-soybean meal based control diet; 2) control diet plus supplemental protease at 5,000 (P5000) protease units (PU)/kg); 3) control plus 10,000 PU/kg protease (P10000); or control plus an enzyme combination containing xylanase, amylase, and protease (XAP) added to achieve protease activity of: 4) 2,500 PU/kg (XAP2500); 5) 5,000 PU/kg (XAP5000); or 6) 10,000 PU/kg (XAP10000). The enzymes in XAP were combined at fixed ratios of 10:1:25 of xylanase:amylase:protease. Data were analyzed by ANOVA and specific orthogonal contrasts between treatments were performed. Addition of xylanase and amylase increased (P < 0.05) the ileal digestibility of protein by 4.2% and 2.1% at XAP5000 and XAP10000, respectively (relative to P5000 and P10000, respectively), exhibiting a plateau after the XAP5000 dose. Increment (P < 0.05) in AME due to protease was evident, particularly in P10000. At the ileal level, XAP reduced (P < 0.05) the flow of insoluble xylose and arabinose, which indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. Protease on its own reduced (P < 0.05) the flow of insoluble arabinose but did not affect the flow of insoluble xylose. XAP reduced (P < 0.05) the pre-cecal flow of insoluble and total glucose and galactose. It was concluded that whereas protease by itself improved nutrient utilization and increased solubilization of NSP components, at the lower dose, a combination of xylanase, amylase, and protease produced effects greater than those of protease alone.

Entrapment of subtilisin in ceramic sol-gel coating for antifouling applications.

Enzymes with antifouling properties are of great interest in developing nontoxic antifouling coatings. A bottleneck in developing enzyme-based antifouling coatings is to immobilize the enzyme in a suitable coating matrix without compromising its activity and stability. Entrapment of enzymes in ceramics using the sol-gel method is known to have several advantages over other immobilization methods. The sol-gel method can be used to make robust coatings, and the aim of this study was to explore if sol-gel technology can be used to develop robust coatings harboring active enzymes for antifouling applications. We successfully entrapped a protease, subtilisin (Savinase, Novozymes), in a ceramic coating using a sol-gel method. The sol-gel formulation, when coated on a stainless steel surface, adhered strongly and cured at room temperature in less than 8 h. The resultant coating was smoother and less hydrophobic than stainless steel. Changes in the coating's surface structure, thickness and chemistry indicate that the coating undergoes gradual erosion in aqueous medium, which results in release of subtilisin. Subtilisin activity in the coating increased initially, and then gradually decreased. After 9 months, 13% of the initial enzyme activity remained. Compared to stainless steel, the sol-gel-coated surfaces with active subtilisin were able to reduce bacterial attachment of both Gram positive and Gram negative bacteria by 2 orders of magnitude. Together, our results demonstrate that the sol-gel method is a promising coating technology for entrapping active enzymes, presenting an interesting avenue for enzyme-based antifouling solutions.

Coupling of epithelial Na+ and Cl- channels by direct and indirect activation by serine proteases.

The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na(+) channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na(+) transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na(+) homeostasis. To explore this process, we examined the coordinated effect of this protease on Na(+) and Cl(-) transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current (I(sc)) was used to measure transport of these ions. The I(sc) exhibited an ENaC-dependent Na(+) component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl(-) component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated I(ENaC) but was without effects on I(CFTR). Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na(+) and Cl(-) transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na(+) and Cl(-) transport in these epithelia.

Thrombolytic effects of Douchi fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo.

BACKGROUND: Today, thrombosis is one of the most widely occurring diseases in modern life. Drugs with thrombolytic functions are the most effective methods in the treatment of thrombosis. Among them, Douchi fibrinolytic enzyme (DFE) is a promising agent. DFE was isolated from Douchi, a typical and popular soybean-fermented food in China, and it can dissolve fibrin directly and efficiently. A strain, Bacillus subtilis LD-8547 produced DFE with high fibrinolytic activity has been isolated in our lab previously. RESULTS: In the study, thrombolytic effect of DFE from Bacillus subtilis LD-8547 was studied in vitro and in vivo systematically. The results showed that DFE played a significant role in thrombolysis and anticoagulation in vitro. And the thrombolytic effects correlated with DFE in a dose-dependent manner. In vivo, the acute toxicity assay showed that DFE had no obvious acute toxicity to mice. Test of carrageenan-induced thrombosis in mice indicated that the DFE significantly prevented tail thrombosis, and arterial thrombosis model test indicated that Douchi fibrinolytic enzyme DFE had thrombolytic effect on carotid thrombosis of rabbits in vivo. Other results in vivo indicated that DFE could increase bleeding and clotting time obviously. CONCLUSIONS: The DFE isolated from Bacillus subtilis LD-8547 has obvious thrombolytic effects in vitro and in vivo. This function demonstrates that this enzyme can be a useful tool for preventing and treating clinical thrombus.

A critical step for JNK activation: isomerization by the prolyl isomerase Pin1.

c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1(-/-) cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.

The serine protease subtilisin suppresses epileptiform activity in rat hippocampal slices and neocortex in vivo.

Serine proteases of the S8A family and those belonging to the subtilase group generate a long-lasting inhibition of hippocampal evoked potentials, which shows little recovery and resembles long-term depression. The present work investigates the effects of subtilisin A on epileptiform activity induced in hippocampal slices. Interictal bursts were generated by perfusion with 4-aminopyridine in magnesium-free medium, whereas ictal bursts were produced by the addition of baclofen. Subtilisin A superfused for 10 min at concentrations of 50 nM and above reduced the duration of ictal bursts, whereas higher concentrations reduced the frequency of interictal activity with little or no recovery, indicating similarity with the long-term depression reported previously. The anti-epileptiform activity was not prevented by inhibitors of phosphatases or several kinases, but the inhibition of ictal activity was selectively reduced by the tyrosine kinase inhibitor genistein. The rho-activated coiled-coil kinase (ROCK) inhibitor Y-27632 had no effect on the suppression of ictal or interictal bursts. Subtilisin applied at nanomolar concentrations to the surface of the cerebral cortex in vivo also suppressed epileptiform spikes induced by bicuculline. It is concluded that serine proteases of the subtilase group are highly potent inhibitors of epileptiform activity, especially ictal bursts, and that tyrosine kinases may be involved in that inhibition. The mechanism of inhibition is different from the long-lasting depression of evoked potentials, which is partly mediated via ROCK.

Steady-state stiffness of utricular hair cells depends on macular location and hair bundle structure.

Spatial and temporal properties of head movement are encoded by vestibular hair cells in the inner ear. One of the most striking features of these receptors is the orderly structural variation in their mechanoreceptive hair bundles, but the functional significance of this diversity is poorly understood. We tested the hypothesis that hair bundle structure is a significant contributor to hair bundle mechanics by comparing structure and steady-state stiffness of 73 hair bundles at varying locations on the utricular macula. Our first major finding is that stiffness of utricular hair bundles varies systematically with macular locus. Stiffness values are highest in the striola, near the line of hair bundle polarity reversal, and decline exponentially toward the medial extrastriola. Striolar bundles are significantly more stiff than those in medial (median: 8.9 µN/m) and lateral (2.0 µN/m) extrastriolae. Within the striola, bundle stiffness is greatest in zone 2 (106.4 µN/m), a band of type II hair cells, and significantly less in zone 3 (30.6 µN/m), which contains the only type I hair cells in the macula. Bathing bundles in media that break interciliary links produced changes in bundle stiffness with predictable time course and magnitude, suggesting that links were intact in our standard media and contributed normally to bundle stiffness during measurements. Our second major finding is that bundle structure is a significant predictor of steady-state stiffness: the heights of kinocilia and the tallest stereocilia are the most important determinants of bundle stiffness. Our results suggest 1) a functional interpretation of bundle height variability in vertebrate vestibular organs, 2) a role for the striola in detecting onset of head movement, and 3) the hypothesis that differences in bundle stiffness contribute to diversity in afferent response dynamics.

Memory impairment in rats by hippocampal administration of the serine protease subtilisin.

Since the serine protease subtilisin has been reported to generate a novel form of long-term depression (LTD) in rat hippocampal slices, the present work was designed to determine whether it has any effect on learning and memory processes. Rats were used to examine the effects of subtilisin, injected directly into the dorsal hippocampus, on task performance in a step-through inhibitory avoidance of a mild footshock. The administration of 100 ng of subtilisin into each hippocampus, immediately after training, was sufficient to induce a detectable learning deficit with a footshock stimulus of 0.5 mA. Higher doses produced dose-related impairments in memory consolidation. These effects were not the result of irreversible toxicity, since rats trained with a higher amplitude footshock (0.75 mA) were able to perform as control animals; therefore, the amnesic effect was not further evident. Furthermore, the administration of subtilisin before avoidance training did not produce any detectable effect on performance during the training or test sessions, indicating that neither acquisition nor consolidation was affected. It is concluded that the post-training administration of a serine protease inhibitor is able to produce robust deficits of memory consolidation consistent with its ability to generate LTD, raising the possibility that related molecules could play physiological or pathological roles in the modulation of learning and memory.