Serum Levels of Coenzyme Q10 in Patients with Multiple System Atrophy

The COQ2 gene encodes an essential enzyme for biogenesis, coenzyme Q10 (CoQ10). Recessive mutations in this gene have recently been identified in families with multiple system atrophy (MSA). Moreover, specific heterozygous variants in the COQ2 gene have also been reported to confer susceptibility to sporadic MSA in Japanese cohorts. These findings have suggested the potential usefulness of CoQ10 as a blood-based biomarker for diagnosing MSA. This study measured serum levels of CoQ10 in 18 patients with MSA, 20 patients with Parkinson’s disease and 18 control participants. Although differences in total CoQ10 (i.e., total levels of serum CoQ10 and its reduced form) among the three groups were not significant, total CoQ10 level corrected by serum cholesterol was significantly lower in the MSA group than in the Control group. Our findings suggest that serum CoQ10 can be used as a biomarker in the diagnosis of MSA and to provide supportive evidence for the hypothesis that decreased levels of CoQ10 in brain tissue lead to an increased risk of MSA.     

A second proliferating cell nuclear antigen loader complex, Ctf18-replication factor C, stimulates DNA polymerase eta activity

Replication factor C (RFC) loads the clamp protein PCNA onto DNA structures. Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dcc1, and Ctf8 and four small RFC subunits, functions as a second proliferating cell nuclear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human cell extracts were assayed for DNA polymerase activity specifically stimulated by Ctf18-RFC in conjunction with PCNA. After several chromatography steps, an activity stimulated by Ctf18-RFC but not by RFC was identified. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis revealed the presence of two DNA polymerases, eta and lambda, in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) eta was responsible for activity. Ctf18-RFC alone stimulated pol eta, and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol eta, as indicated by co-precipitation in human cells. We propose that this novel loader-DNA polymerase interaction allows DNA replication forks to overcome interference by various template structures, including damaged DNA and DNA-protein complexes that maintain chromosome cohesion.     

CO2-dependent migration and relocation of LCIB,a pyrenoid-peripheral protein in Chlamydomonas reinhardtii

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO₂-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO₂-limiting conditions (low-CO₂ [LC] and very low-CO₂ [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO₂-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO₂-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3–15 µM CO₂, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO₂. Furthermore, LCIB relocated toward the pyrenoid at 2.6–3.4 µM CO₂, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO₂. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO₂ concentration with ∼7 µM as the boundary.

Algal chloroplast proteins undergo localization changes in response to CO2 concentrations, reflecting their physiological function in survival under fluctuating CO2 environments.     

Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18(-/-) cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein-protein interaction and PCNA monoubiquitination.     

Soluble branched beta-(1,4)glucans from Acetobacter species show strong activities to induce interleukin-12 in vitro and inhibit T-helper 2 cellular response with immunoglobulin E production in vivo

An extracellular polysaccharide, AC-1, produced by Acetobacter polysaccharogenes is composed of beta-(1,4)glucan with branches of glucosyl residues. We found that AC-1 showed a strong activity to induce production of interleukin-12 P40 and tumor necrosis factor-alpha by macrophage cell lines in vitro. Cellulase treatment completely abolished the activity of AC-1 to induce tumor necrosis factor-alpha production by macrophages, whereas treatment of AC-1 with polymyxin B or proteinase did not affect the activity. Results of experiments using toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of AC-1. In vivo administration of AC-1 significantly reduced the serum levels of ovalbumin (OVA)-specific IgE and interleukin-4 production by T cells in response to OVA in mice immunized with OVA. AC-1, a soluble branched beta-(1,4)glucan may be useful in prevention and treatment of allergic disorders With IgE production.     

Rib72, a conserved protein associated with the ribbon compartment of flagellar A-microtubules and potentially involved in the linkage between outer doublet microtubules

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an approximately 96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.     

DC3, the smallest subunit of the Chlamydomonas flagellar outer dynein arm-docking complex,is a redox-sensitive calcium-binding protein

The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. The Chlamydomonas ODA-DC contains three proteins; loss of any one prevents normal assembly of the outer arm, leading to a slow, jerky swimming phenotype. We showed previously that the smallest ODA-DC subunit, DC3, has four EF-hands (Casey, D. M., Inaba, K., Pazour, G. J., Takada, S., Wakabayashi, K., Wilkerson, C. G., Kamiya, R., and Witman, G. B. (2003) Mol. Biol. Cell 14, 3650-3663). Two of the EF-hands fit the consensus pattern for calcium binding, and one of these contains two cysteine residues within its binding loop. To determine whether the predicted EF-hands are functional, we purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of dithiothreitol and Mg2+. The protein bound one calcium ion with an affinity (Kd) of approximately 1 x 10-5 m. Calcium binding was observed only in the presence of dithiothreitol and thus is redox-sensitive. DC3 also bound Mg2+ at physiological concentrations but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium binding activity of the bacterially expressed protein. To investigate the role of the EF-hands in vivo, we transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the Glu to Gln mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains unknown.     

Regulation of the Gts1p level by the ubiquitination system to maintain metabolic oscillations in the continuous culture of yeast

Yeast cells exhibit sustained ultradian oscillations of energy metabolism in coupling with cell cycle and stress resistance oscillations in continuous culture. We have reported that the rhythmic expression of Gts1p is important for the maintenance of ultradian rhythms. Structurally, Gts1p contains sequence motifs similar to N-degron and the ubiquitin association domain, raising the possibility that the Gts1p level is regulated by degradation via ubiquitination. When the lysine residue at the putative ubiquitination site of the N-degron was substituted with arginine, both the protein level and half-life of mutant Gts1p increased. During continuous culture, the protein level of the mutant Gts1p was elevated and did not fluctuate, leading to the disappearance of metabolic oscillation within a day. Furthermore, using three Gts1ps containing mutations in the ubiquitin association domain, we showed that the lower the binding activity of the mutant Gts1ps for polyubiquitin in vitro, the higher the protein level in vivo. Expression of the mutant Gts1ps in the continuous culture resulted in an increase in Gts1p and early loss of the oscillation. Therefore, Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiquitinated Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillations.