Nitrooxyethylation reverses the healing-suppressant effect of Ibuprofen

Nonsteroidal antiinflammatory drugs like ibuprofen impede tissue repair by virtue of retarding inflammation. The present study was undertaken to explore if linking of nitrooxyethyl ester to ibuprofen reverses its healing-depressant propensity. Nitrooxyethyl ester of ibuprofen (NOE-Ibu) was synthesized in our laboratory through a well-established synthetic pathway. NOE-Ibu was screened for its influence on collagenation, wound contraction and epithelialization phases of healing, and scar size of healed wound in three wound models, namely, incision, dead space, and excision wounds. Besides, its influence on the oxidative stress (levels of GSH and TBARS) was also determined in 10-day-old granulation tissue. NOE-Ibu was further screened for its antiinflammatory activity in rat paw edema model. NOE-Ibu promoted collagenation (increase in breaking strength, granulation weight, and collagen content), wound contraction and epithelialization phases of healing. NOE-Ibu also showed a significant antioxidant effect in 10-day-old granulation tissue as compared to ibuprofen. Results vindicate that the esterification of ibuprofen with nitrooxyethyl group reverses the healing-suppressant effect of ibuprofen. The compound also showed equipotent antiinflammatory activity as ibuprofen.     

Reovirus mu1 structural rearrangements that mediate membrane penetration

Membrane penetration by nonenveloped reoviruses is mediated by the outer-capsid protein, mu1 (76 kDa). Previous evidence has suggested that an autolytic cleavage in mu1 allows the release of its N-terminally myristoylated peptide, mu1N (4 kDa), which probably then interacts with the target-cell membrane. A substantial rearrangement of the remaining portion of mu1, mu1C (72 kDa), must also have occurred for mu1N to be released, and some regions in mu1C may make additional contacts with the membrane. We describe here a particle-free system to study conformational rearrangements of mu1. We show that removal of the protector protein sigma3 is not sufficient to trigger rearrangement of free mu1 trimer and that free mu1 trimer undergoes conformational changes similar to those of particle-associated mu1 when induced by similar conditions. The mu1 rearrangements require separation of the mu1 trimer head domains but not the mu1N/C autocleavage. We have also obtained a relatively homogeneous form of the structurally rearranged mu1 (mu1*) in solution. It is an elongated monomer and retains substantial alpha-helix content. We have identified a protease-resistant approximately 23-kDa fragment of mu1*, which contains the largely alpha-helical regions designated domains I and II in the conformation of mu1 prior to rearrangement. We propose that the mu1 conformational changes preceding membrane penetration or disruption during cell entry involve (i) separation of the beta-barrel head domains in the mu1 trimer, (ii) autolytic cleavage at the mu1N/C junction, associated with partial unfolding of mu1C and release of mu1N, and (iii) refolding of the N-terminal helical domains of mu1C, with which mu1N was previously complexed, accompanied by dissociation of the mu1 trimer.     

Reovirus outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria

The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus micro1 protein, encoded by the M2 gene, was sufficient to induce apoptosis in transfected cells. We also found that micro1 localized to lipid droplets, endoplasmic reticulum, and mitochondria in both transfected cells and infected cells. Two small regions encompassing amphipathic alpha-helices within a carboxyl-terminal portion of micro1 were necessary for efficient induction of apoptosis and association with lipid droplets, endoplasmic reticulum, and mitochondria in transfected cells. Induction of apoptosis by micro1 and its association with lipid droplets and intracellular membranes in transfected cells were abrogated when micro1 was coexpressed with sigma3, with which it is known to coassemble. We propose that micro1 plays a direct role in the induction of apoptosis in infected cells and that this property may relate to the capacity of micro1 to associate with intracellular membranes. Moreover, during reovirus infection, association with sigma3 may regulate apoptosis induction by micro1.     

Specificity of the myotubularin family of phosphatidylinositol-3-phosphatase is determined by the PH/GRAM domain

Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease.     

Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1

Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.     

Acetone production in solventogenic <Emphasis Type="Italic">Clostridium</Emphasis> species: new insights from non-enzymatic decarboxylation of acetoacetate

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO₂, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc ⁻ mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation. Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol (ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to enhance selectivity for butanol production.     

Effect of Dehydrozingerone, a half analog of curcumin on dexamethasone-delayed wound healing in albino rats

Oxidative stress is triggered by the wound which results in the production of reactive oxygen species (ROS), thereby delaying normal wound repair. Therefore, it is important to reduce the level of ROS to improve healing. A known antioxidant, dehydrozingerone (DHZ) was synthesized and selected for the study. The authors aimed to investigate the wound healing action of topical (100 mg/wound) and systemic (100 mg/kg, p. o.). DHZ on different wound models in normal and dexamethasone (DEX)-suppressed healing. Topical DHZ showed a significant (P < 0.05) rise in tensile strength when compared to control in normal healing. Significant (P < 0.05) wound closure was observed from 3 to 9 days in DHZ oral and gel treated groups. There was a significant (P < 0.05) rise in hydroxyproline content with the DHZ treated groups when compared to control. Systemic DHZ exhibited a significant (P < 0.05) increase in lysyl oxidase (LO) levels of 3.73 ± 0.15 nmol of H(2)O(2) when compared to control. In DEX-suppressed healing, showed good pro-healing activity with respect to the parameters mentioned above. DHZ treatment exhibited a parabolic dose response of ROS inhibition with a plateau effect at 75 μM. There was a steady and constant increase in the % NO inhibition with increasing doses of DHZ. Oral DHZ is effective in accelerating the healing process in both normal and dexamethasone-suppressed wounds. Our study suggests that DHZ (half analog of curcumin) supplementation reduces the steroid-induced delay in wound healing.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.