Spontaneous dimerization of titin protein Z1Z2 domains induces strong nanomechanical anchoring

Muscle elasticity strongly relies on the mechanical anchoring of the giant protein titin to both the sarcomere M-band and the Z-disk. Such strong attachment ensures the reversible dynamics of the stretching-relaxing cycles determining the muscle passive elasticity. Similarly, the design of biomaterials with enhanced elastic function requires experimental strategies able to secure the constituent molecules to avoid mechanical failure. Here we show that an engineered titin-mimicking protein is able to spontaneously dimerize in solution. Our observations reveal that the titin Z1Z2 domains are key to induce dimerization over a long-range distance in proteins that would otherwise remain in their monomeric form. Using single molecule force spectroscopy, we measure the threshold force that triggers the noncovalent transition from protein dimer to monomer, occurring at ～700 piconewtons. Such extremely high mechanical stability is likely to be a natural protective mechanism that guarantees muscle integrity. We propose a simple molecular model to understand the force-induced dimer-to-monomer transition based on the geometric distribution of forces occurring within a dimeric protein under mechanical tension.     

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.     

Synthesis and structure-activity relationships of selective norepinephrine reuptake inhibitors (sNRI) with a heterocyclic ring constraint

The design, synthesis and SAR of a series of heterocyclic ring-constrained norepinephrine reuptake inhibitors are described. As racemates, the best compounds compare favorably with atomoxetine (IC(50)'s<10 nM) in potency at the transporter.     

Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePP_i), a potent mineralization inhibitor, to phosphate (P_i). By the controlled hydrolysis of ePP_i, TNAP maintains the correct ratio of P_i to ePP_i and therefore enables normal skeletal and dental calcification. In other areas of the body low ePP_i levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePP_i. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.     

The anticancer impacts of free and liposomal caffeic acid phenethyl ester (CAPE) on melanoma cell line (A375)

The deadliest type of skin cancer, malignant melanoma, is also the reason for the majority of skin cancer-related deaths. The objective of this article was to investigate the efficiency of free caffeic acid phenethyl ester (CAPE) and liposomal CAPE in inducing apoptosis in melanoma cells (A375) in in vitro. CAPE was loaded into liposomes made up of hydrogenated soybean phosphatidylcholine, cholesterol, and 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], and their physicochemical properties were assessed. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed for comparing the cytotoxicity of free CAPE and liposomal CAPE at dosages of 10, 15, 25, 50, 75 and the highest dose of 100 μg/mL for period of 24 and 48 h on A375 cell line to calculate IC50. Apoptosis and necrosis were evaluated in A375 melanoma cancer cells using flow cytometry. Atomic force microscopy was utilized to determine the nanomechanical attributes of the membrane structure of A375 cells. To determine whether there were any effects on apoptosis, the expression of PI3K/AKT1 and BAX/BCL2 genes was analyzed using the real-time polymerase chain reaction technique. According to our results, the maximum amount of drug release from nanoliposomes was determined to be 91% and the encapsulation efficiency of CAPE in liposomes was 85.24%. Also, the release of free CAPE was assessed to be 97%. Compared with liposomal CAPE, free CAPE showed a greater effect on reducing the cancer cell survival after 24 and 48 h. Therefore, IC50 values of A375 cells treated with free and liposomal CAPE were calculated as 47.34 and 63.39 μg/mL for 24 h. After 48 h of incubation of A375 cells with free and liposomal CAPE, IC50 values were determined as 30.55 and 44.83 μg/mL, respectively. The flow cytometry analysis revealed that the apoptosis induced in A375 cancer cells was greater when treated with free CAPE than when treated with liposomal CAPE. The highest nanomechanical changes in the amount of cell adhesion forces, and elastic modulus value were seen in free CAPE. Subsequently, the greatest decrease in PI3K/AKT1 gene expression ratio occurred in free CAPE.     

Cep78 controls centrosome homeostasis by inhibiting EDD‐DYRK2‐DDB1VprBP

The centrosome plays a critical role in various cellular processes including cell division and cilia formation, and deregulation of centrosome homeostasis is a hallmark feature of many human diseases. Here, we show that centrosomal protein of 78 kDa (Cep78) localizes to mature centrioles and directly interacts with viral protein R binding protein (VprBP). Although VprBP is a component of two distinct E3 ubiquitin ligases, EDD‐DYRK2‐DDB1^VprBP and CRL4^VprBP, Cep78 binds specifically to EDD‐DYRK2‐DDB1^VprBP and inhibits its activity. A pool of EDD‐DYRK2‐DDB1^VprBP is active at the centrosome and mediates ubiquitination of CP110, a novel centrosomal substrate. Deregulation of Cep78 or EDD‐DYRK2‐DDB1^VprBP perturbs CP110 ubiquitination and protein stability, thereby affecting centriole length and cilia assembly. Mechanistically, ubiquitination of CP110 entails its phosphorylation by DYRK2 and binding to VprBP. Cep78 specifically impedes the transfer of ubiquitin from EDD to CP110 without affecting CP110 phosphorylation and binding to VprBP. Thus, we identify Cep78 as a new player that regulates centrosome homeostasis by inhibiting the final step of the enzymatic reaction catalyzed by EDD‐DYRK2‐DDB1^VprBP.