Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis

The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.Translational regulation is a fundamental mechanism used to control the accumulation of key proteins in a large variety of biogenetic and physiological processes in both prokaryotic and eukaryotic cells (20, 23). Translational autoregulation is a particular form of regulation exerted by the protein being translated. It is a well-established control mechanism for bacteriophage and prokaryotic systems (15), and it has also been reported in eukaryotes (4). Usually, the newly synthesized protein binds to its own mRNA to repress translation (20). However, repression can also be exerted by nascent chains interacting with the ribosome (49).Translational autoregulation also occurs in semiautonomous eukaryotic organelles of ancestral bacterial origin, namely, mitochondria and chloroplasts. During evolution, these organelles have retained a few genes in their own genomes, which are transcribed within the organelle, and the mRNAs are translated on organellar ribosomes. Most proteins synthesized within the organelles are part of large multimeric enzyme complexes devoted to energy production. These complexes are formed by subunits of dual genetic origin, nuclear and organellar, and assemble in the organellar membranes. Interestingly, intraorganellar translation of certain subunits has been proposed to be regulated by the availability of their assembly partners (1, 39, 54, 55). A distinctive characteristic of these systems is the involvement of ternary factors, mRNA-specific translational activators whose availability would be regulated by the specific gene products. The players and mechanisms involved remain largely unknown.We have focused on the characterization, in the yeast Saccharomyces cerevisiae, of an assembly-controlled translational regulatory system that operates during the biogenesis of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain. The three subunits forming the COX catalytic core (1, 2, and 3) are encoded in the mitochondrial DNA (mtDNA), and the remaining eight subunits are encoded in the nuclear DNA. Subunits 1 and 2 coordinate the heme A and copper prosthetic groups of the enzyme. COX biogenesis requires the assistance of a large number of ancillary factors acting at all the levels of the process (11). COX assembly is thought to be linear, consisting of the sequential addition of subunits to an initial seed formed by the mtDNA-encoded subunit 1 (Cox1) in both mammalian and yeast cells (11).The concerted accumulation of COX subunits is regulated by posttranslational degradation of most unassembled Cox1 and the other highly hydrophobic core subunits (27). Recently, we along with others have proposed an additional level of regulation, namely, an assembly-controlled synthesis of Cox1 (1, 2, 39, 56). In S. cerevisiae, COX1 mRNA translation is under the control of Mss51 and Pet309 (8, 30). Mss51 is a key element of the regulatory system. Mss51 acts on the 5′ untranslated region (UTR) of COX1 mRNA to promote translation initiation (39, 56) and additionally acts on a target in the protein coding sequence of COX1 mRNA, perhaps to promote elongation (39). Mss51 and newly synthesized Cox1 form a transient complex (2, 39) that is stabilized by Cox14 (2). We have postulated that these interactions downregulate Cox1 synthesis when COX assembly is impaired by trapping Mss51 and limiting its availability for COX1 mRNA translation (2). According to this model, the release of Mss51 from the ternary complex and its availability for Cox1 synthesis probably occur when Cox1 acquires its prosthetic groups or interacts with other COX subunits, a step possibly catalyzed by Shy1, a protein involved in maturation and/or assembly of Cox1 (2, 10, 34). Coa1 could also participate in Cox1 maturation and stabilize the ternary Cox1/Mss51/Cox14 complex until it interacts with Shy1 (34, 40). Further studies are required to understand how Mss51 is recycled from its posttranslational function to become available for COX1 mRNA translation and to fully clarify how this regulatory mechanism operates.In this study, we have analyzed protein-interacting partners of Mss51 in the wild type and a collection of COX assembly mutants. We found that the native molecular weight (MW) of Mss51 is dependent on both the status of COX assembly and the synthesis of Cox1. The mitochondrial Hsp70 (mtHsp70) chaperone Ssc1 interacts with Mss51 and with several high-molecular weight Mss51-containing complexes involving the COX1 mRNA translational apparatus, Cox1, and several Cox1 assembly factors. Mutants defective in Cox1 maturation or in other aspects of COX biogenesis accumulate distinct ratios of these complexes. In this way, Cox1 regulates its own translation through the action of Mss51 and Ssc1.     

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels     

In vivo degradation behavior of photo-cross-linked star-poly(epsilon-caprolactone-co-D,L-lactide) elastomers

We have recently reported on the preparation of biodegradable elastomers through photo-cross-linking acrylated star-poly(epsilon-caprolactone-co-D,L-lactide). In this paper we assess the change in their physical properties during in vivo degradation in rats after subcutaneous implantation over a 12 week period. These parameter changes were compared to those observed in vitro. Two different cross-link densities were examined, representing the range from a high Young's modulus to a low Young modulus. Elastomers having a high cross-link density exhibited degradation behavior consistent with a surface erosion mechanism, and degraded at the same rate in vivo as observed in vitro. Young's modulus and the stress at break of these elastomers decreased linearly with the degradation time, while the strain at break decreased slowly. Elastomers having a low cross-link density exhibited a degradation mechanism consistent with bulk erosion. Young's modulus and the stress at break of these elastomers decreased slowly initially, followed by a marked increase in mechanical strength loss after 4 weeks. The elastomers were well tolerated by the rats over the 12 week period in vivo.     

Continuous monitoring of predator control operations at landscape scale

Summary   Exotic predators are considered pests to wildlife and agriculture, requiring predator-control programs. Effective monitoring of predator-control operations is essential to justify their considerable cost, but often impossible in practice. The difficulties are especially severe if the target species is small and wide-ranging, and the area to be protected is inaccessible and/or extensive. A convenient model predator of this type, the feral Ferret ( Mustela furo ), is subject to control on farmland in New Zealand. We monitored the distribution of Ferrets over 2400 ha in the central North Island, before and throughout a standard control operation by professional trappers. We used 24 units of a new automated monitoring device, the Scentinel, set in a grid at 1 per 100 ha. Over 11 weeks (11 February to 29 April 2005, 1718 trap nights), we recorded 1559 visits by small mammals, including 198 by Ferrets. By the end of the 4th week, Ferrets had been detected at 17 of 24 sites. Removal of Ferrets from the study area by contractors began during the 5th week, and was reflected in significant declines in the number of Ferret visits recorded per day ( P  = 0.008) and the number of sites visited ( P  = 0.021). Analysis of our extensive repeat-survey data by site-occupancy methods confirmed these trends in greater detail, while also allowing for variation in detectability.     

Mechanical properties of individual focal adhesions probed with a magnetic microneedle

A permanent magnetic microneedle was developed to apply tensional forces to integrin receptors via ligand-coated magnetic microbeads while optically analyzing the mechanical properties of individual focal adhesions. Force application (130 pN for 3 s) through activated beta1 integrins produced less bead displacement than when unligated integrins were stressed. This strengthening response differed markedly on a bead-by-bead basis, correlated directly with local focal adhesion assembly, and was similar when analyzed at 4 degrees C, indicating that it was due to passive material properties of the cell. Viscoelastic analysis clarified that recruitment of focal adhesion proteins increased the local elastic stiffness of the adhesion complex without changing its viscous behavior. These data indicate that individual focal adhesions exhibit distinct mechanical properties that depend upon local focal adhesion assembly, and that these local variations in micromechanics can be detected and analyzed within living cells using the permanent magnetic microneedle technique.     

Ultrastructural evidence of autoinfection in the gills of Atlantic cod Gadus morhua infected with Loma sp. (phylum Microsporidia)

Infection by a microsporidian of the genus Loma was found in gills of cod Gadus morhua. Xenomas contained parasites in multiple stages of development. Some spores looked empty and had everted polar tubes, which were either straight or coiled. These polar tubes were scattered throughout the xenoma cytoplasm, and some of them pierced the plasma membrane. Those outside of the xenoma penetrated neighboring cells, including blood cells. These observations suggest that a mechanism of autoinfection could occur in blood cells and gill tissue, perpetuating the disease in the host.     

Activated human dendritic cells express inducible cyclo-oxygenase and synthesize prostaglandin E2 but not prostaglandin D2

Prostaglandins (PG) are well known lipid mediators with important immunoregulatory properties. While exogenous PGE2 has the ability to modulate the function and maturation of antigen presenting cells, such as dendritic cells (DC), it is not clear whether human DC have the capacity to synthesize PGE2 and other prostaglandins themselves. We therefore examined the expression of inducible cyclo-oxygenase (COX-2) by monocyte derived DC and the production of PGE2 and PGD2. Both monocyte derived DC and freshly isolated blood myeloid DC expressed little COX-2 constitutively, though COX-2 expression was rapidly but transiently upregulated in response to lipopolysaccharide stimulation. COX-2 mRNA was detectable within 1 h of LPS exposure, peaked at 4-6 h, and rapidly declined thereafter. COX-2 expression was accompanied by DC synthesis of PGE2, with peak levels present at 6-18 h post-stimulation. In contrast, PGD2 synthesis was not detected at any time point. When DC were activated with LPS in the presence of nimesulide, a COX-2 selective inhibitor, IL-10 synthesis was inhibited, indicating that endogenous prostaglandins regulate DC cytokine production. PGE2 production by DC may therefore modulate DC and T-cell function, thereby shaping the character of the immune response.     

New hominin first metatarsal (SK 1813) from Swartkrans

A recently recognized hominin hallucal metatarsal, SK 1813, from Swartkrans bears a suite of primitive and derived traits. Comparisons with extant apes, modern humans, SKX 5017, and Stw 562 reveals similar morphology in all three fossils and that these early hominins, while bipedal, possessed a unique toe-off mechanism. The implications of this are that both primitive and derived traits must be used to establish the total biomechanical pattern.