Crystal structure of the Ube2K/E2-25K and K48-linked di-ubiquitin complex provides structural insight into the mechanism of K48-specific ubiquitin chain synthesis.

Ubiquitin-conjugating enzymes (E2) form thioester bonds with ubiquitin (Ub), which are subsequently transferred to target proteins for cellular progress. Ube2K/E2-25K (a class II E2 enzyme) contains a C-terminal ubiquitin-associated (UBA) domain that has been suggested to control ubiquitin recognition, dimerization, or poly-ubiquitin chain formation. Ube2K is a special E2 because it synthesizes K48-linked poly-ubiquitin chains without E3 ubiquitin ligase. We found that a novel interaction between the acceptor di-Ub (Ub2) and the auxiliary Ube2K promotes the discharging reaction and production of tri-Ub (Ub3), probably by guiding and positioning the K48 (in the distal Ub) of the acceptor Ub2 in the active site. We also determined the crystal structure of Ube2K-Ub2 at 2.47 Šresolution. Based on our structural and biochemical data, we proposed a structural model of Ub3 synthesis by Ube2K without E3.

Structural and biochemical insights into inhibition of human primase by citrate.

The eukaryotic primase/polymerase complex synthesizes approximately 107 primers, one per Okazaki fragment, during the replication of mammalian chromosomes, which contain 109 base pairs. Primase catalyzes the synthesis of a short RNA segment to a single-stranded DNA template. Primase is important in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA primer. The primase subcomplex is composed of a small catalytic subunit (p49), and a large accessory subunit (p58). Priming mechanisms remain poorly understood, although large numbers of structures of archaeal and eukaryotic p49 and/or p58 as well as structures of bacterial enzymes have been determined. In this study, we determined the structure of human p49 at 2.2 Šresolution with citrate in its inactive forms. Dibasic citrate was bound at the nucleotide triphosphate (NTP) ß, γ-phosphate binding site through nine hydrogen bonds. We also measured the dissociation constant of citrate and NTPs. We further demonstrated that the p49 activity is regulated by pH and citrate, which was not previously recognized as a key regulator of DNA replication. We propose that the citrate inhibits the primase and regulates DNA replication at the replication fork.

Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima.

Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. The structure and function of the ATPase and protease domains of FtsH have been previously characterized while the role of the FtsH periplasmic domain has not clearly identified. Here, we report the 1.5-1.95 Å resolution crystal structures of the Thermotoga maritima FtsH periplasmic domain (tmPD) and describe the dynamic features of tmPD oligomerization.

Structural mechanism underlying regulation of human EFhd2/Swiprosin-1 actin-bundling activity by Ser183 phosphorylation.

EF-hand domain-containing protein D2/Swiprosin-1 (EFhd2) is an actin-binding protein mainly expressed in the central nervous and the immune systems of mammals. Intracellular events linked to EFhd2, such as membrane protrusion formation, cell adhesion, and BCR signaling, are triggered by the association of EFhd2 and F-actin. We previously reported that Ca2+ enhances the F-actin-bundling ability of EFhd2 through maintaining a rigid parallel EFhd2-homodimer structure. It was also reported that the F-actin-bundling ability of EFhd2 is regulated by a phosphorylation-dependent mechanism. EGF-induced phosphorylation at Ser183 of EFhd2 has been shown to inhibit F-actin-bundling, leading to irregular actin dynamics at the leading edges of cells. However, the underlying mechanism of this inhibition has remained elusive. Here, we report the crystal structure of a phospho-mimicking mutant (S183E) of the EFhd2 core domain, where the actin-binding sites are located. Although the overall structure of the phospho-mimicking mutant is similar to the one of the unphosphorylated form, we observed a conformational transition from ordered to disordered structure in the linker region at the C-terminus of the mutant. Based on our structural and biochemical analyses, we suggest that phosphorylation at Ser183 of EFhd2 causes changes in the local conformational dynamics and the surface charge distribution of the actin-binding site, resulting in a re-coordination of the actin-binding sites in the dimer structure and a reduction of F-actin-bundling activity without affecting the F-actin-binding capacity.

Crystal structure of the catalytic domain of Clostridium perfringens neuraminidase in complex with a non-carbohydrate-based inhibitor, 2-(cyclohexylamino)ethanesulfonic acid.

Anti-bacterial and anti-viral neuraminidase agents inhibit neuraminidase activity catalyzing the hydrolysis of terminal N-acetylneuraminic acid (Neu5Ac) from glycoconjugates and help to prevent the host pathogenesis that lead to fatal infectious diseases including influenza, bacteremia, sepsis, and cholera. Emerging antibiotic and drug resistances to commonly used anti-neuraminidase agents such as oseltamivir (Tamiflu) and zanamivir (Relenza) have highlighted the need to develop new anti-neuraminidase drugs. We obtained a serendipitous complex crystal of the catalytic domain of Clostridium perfringens neuraminidase (CpNanICD) with 2-(cyclohexylamino)ethanesulfonic acid (CHES) as a buffer. Here, we report the crystal structure of CpNanICD in complex with CHES at 1.24 Å resolution. Amphipathic CHES binds to the catalytic site of CpNanICD similar to the substrate (Neu5Ac) binding site. The 2-aminoethanesulfonic acid moiety and cyclohexyl groups of CHES interact with the cluster of three arginine residues and with the hydrophobic pocket of the CpNanICD catalytic site. In addition, a structural comparison with other bacterial and human neuraminidases suggests that CHES could serve as a scaffold for the development of new anti-neuraminidase agents targeting CpNanI.

Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

Structural insights into the interaction of p97 N-terminus domain and VBM in rhomboid protease, RHBDL4.

RHBDL4 is an active rhomboid that specifically recognizes and cleaves atypical, positively charged transmembrane endoplasmic reticulum-associated degradation (ERAD) substrates. Interaction of valosin-containing protein (p97/VCP) and RHBDL4 is crucial to retrotranslocate polyubiquitinated substrates for ERAD pathway. Here, we report the first complex structure of VCP-binding motif (VBM) with p97 N-terminal domain (p97N) at 1.88 Šresolution. Consistent with p97 adaptor proteins including p47-ubiquitin regulatory X (UBX), gp78-VCP-interacting motif (VIM), OTU1-UBX-like element, and FAF1-UBX, RHBDL4 VBM also binds at the interface between the two lobes of p97N. Notably, the RF residues in VBM are involved in the interaction with p97N, showing a similar interaction pattern with that of FPR signature motif in the UBX domain, although the directionality is opposite. Comparison of VBM interaction with VIM of gp78, another α-helical motif that interacts with p97N, revealed that the helix direction is inversed. Nevertheless, the conserved arginine residues in both motifs participate in the majority of the interface via extensive hydrogen bonds and ionic interactions with p97N. We identified novel VBM-binding mode to p97N that involves a combination of two types of p97-cofactor specificities observed in the UBX and VIM interactions. This highlights the induced fit model of p97N interdomain cleft upon cofactor binding to form stable p97-cofactor complexes. Our mutational and biochemical analyses in defining the specific interaction between VBM and p97N have elucidated the importance of the highly conserved VBM, applicable to other VBM-containing proteins. We also showed that RHBDL4, ubiquitins, and p97 co-operate for efficient substrate dislocation.

Hepatocellular carcinoma risk in chronic hepatitis B virus-infected compensated cirrhosis patients with low viral load.

UNLABELLED: Controversy exists about whether antiviral therapy (AVT) should be recommended for compensated cirrhosis patients with chronic hepatitis B virus (HBV) infection and detectable, but low, serum HBV-DNA levels. A retrospective cohort of 385 treatment-naïve, HBV-related compensated cirrhosis patients (mean age: 51.1 ± 9.7 years; 66% male) with low HBV-DNA levels (<2,000 IU/mL) was assessed for the development of hepatocellular carcinoma (HCC). During a median of 5.6 years of follow-up, HCC had developed in 37 (9.6%) patients. The 5-year cumulative HCC incidence rate was 2.2%, 8.0%, and 14.0% for patients with undetectable HBV DNA (<12 IU/mL), low HBV-DNA levels plus normal alanine aminotransferase (ALT) levels, and low HBV-DNA levels plus elevated ALT levels at baseline (P = 0.011). During follow-up, 71 patients maintained undetectable HBV-DNA levels, and 126 experienced HBV-DNA elevation over 2,000 IU/mL. AVT was initiated in 77 patients. In patients without AVT, the 5-year cumulative HCC incidence rates were 13.3%, 8.8%, and 1.4% for those who experienced HBV-DNA elevation, those who maintained detectable, but low, HBV-DNA levels, and those who maintained undetectable HBV-DNA levels, respectively. The 5-year cumulative HCC incidence rate was 5.9% for patients who started AVT; longer AVT duration and longer complete virological response (<12 IU/mL) duration was associated with lower HCC risk. CONCLUSION: Compensated cirrhosis patients with detectable, but low, viral load were not at low risk for HCC, and AVT was associated with lower HCC risk, suggesting that prompt AVT should be considered for these patients.

Hepatocellular Carcinoma Risk of Compensated Cirrhosis Patients with Elevated HBV DNA Levels according to Serum Aminotransferase Levels.

Sometimes, hepatitis B virus (HBV)-related cirrhotic patients with normal aminotransferase levels are closely followed-up for the elevation of aminotransferase levels instead of prompt antiviral therapy (AVT). We analyzed the long-term hepatocellular carcinoma (HCC) risk according to the aminotransferase levels in a retrospective cohort of 1,468 treatment-naïve, HBV-related, compensated cirrhosis patients with elevated HBV DNA levels (≥ 2,000 IU/mL). Based on aminotransferase levels, patients were categorized into normal (< 40 U/L, n = 364) and elevated group (≥ 40 U/L, n = 1,104). During a median of 5.3 yr of follow-up (range: 1.0-8.2 yr), HCC developed in 296 (20%) patients. The 5-yr cumulative HCC incidence rate was higher in patients with elevated aminotransferase level, but was not low in normal aminotransferase level (17% vs. 14%, P = 0.004). During the follow-up, 270/364 (74%) patients with normal aminotransferase levels experienced elevation of aminotransferase levels, and AVT was initiated in 1,258 (86%) patients. Less patients with normal aminotransferase levels received AVT (70% vs. 91%, P < 0.001) and median time to start AVT was longer (17.9 vs. 2.4 months, P < 0.001). AVT duration was an independent factor associated with HCC, and median duration of AVT was shorter (4.0 vs. 2.6 yr, P < 0.001) in patients with normal aminotransferase levels. The HCC risk of compensated cirrhosis patients with normal aminotransferase level is not low, and AVT duration is associated with lowered HCC risk, indicating that prompt AVT should be strongly considered even for those with normal aminotransferase levels.

Hypolipidemic and antiinflammation activities of fermented soybean fibers from meju in C57BL/6 J mice.

Meju, a naturally fermented soy block used to produce soy paste and soy sauce in Korea, is suggested to exhibit hypolipidemic and antiinflammatory activities; however, its mechanisms of action are elusive. Here, we report that the water-soluble fibers but not the amino acids and peptides from meju exhibited hypolipidemic activity in vivo. Feeding of fermented soybean fibers (FSF) from meju reduced plasma cholesterol, triglyceride, adipocyte size, and hepatic lipid accumulation in C57BL/6 J mice. FSF treatment reduced HMG-CoA reductase expression, whereas the expression of genes in the fatty acid uptake and subsequent beta-oxidation were significantly induced in the livers. Hepatic lipogenic genes, including Srebp1c and Lxrα, were unaltered. Feeding with the fermented soybean peptides and amino acids (FSPA) induced the expression of lipogenic genes, which may have canceled the induction of low-density lipoprotein receptor and Cyp7a1 gene expressions in FSPA livers. The plasma concentrations of C-reactive protein, TNF-α, and interlukin-6 were significantly reduced in the FSF, FSPA, and meju groups compared with the control groups, suggesting that both of the fibers and peptides/amino acids from meju may be beneficial. These findings suggest that soluble fibers from meju are critical hypolipidemic components that regulate hepatic gene expressions and reduce proinflammatory cytokines in vivo.

Association between viral hepatitis B infection and halitosis.

OBJECTIVE: Oral malodor can be increased in breath of liver patients. However, no study has been performed for the association between volatile sulfur compounds (VSCs) and viral hepatitis. The aim of the present study was to determine the relationship between viral hepatitis and VSCs. METHODS: This study analyzed 182 subjects and measured hydrogen sulfide (H2S), methyl mercaptan (CH3SH) and dimethyl sulfide [(CH3)2S] using the OralChroma(®). Hepatitis type B was evaluated. Periodontal health was assessed using the Community Periodontal Index (CPI) and bleeding on probing (BOP). Tongue coating score (TCS) was evaluated. Multiple logistic regression analyses were conducted to evaluate the relationship. RESULTS: Viral hepatitis had an elevated odds of dimethyl sulfide defined halitosis (OR = 9.22, 95% CI = 2.08-40.95) after controlling for age, gender, alcohol consumption, current smoking, periodontitis, BOP, TCS and tongue brushing habit. The magnitude of the association between viral hepatitis and VSCs defined halitosis attenuated with adjustment of mediators (alcohol consumption, periodontitis, BOP, TCS and tongue brushing habit for hydrogen sulfide defined halitosis; periodontitis, TCS and tongue brushing habit for methyl mercaptan defined halitosis; tongue brushing habit for dimethyl sulfide defined halitosis). CONCLUSIONS: Findings of this study suggest that viral hepatitis may be associated with methyl mercaptan defined halitosis.

A missense mutation (c.1963A

Serum Ca(++) levels play important roles in the humoral immunity. The aim of this study was to detect quantitative trait loci and the associated positional candidate genes affecting baseline serum Ca(++) concentrations. A genome-wide association study was conducted in an F(2) intercross population between Landrace and Korean native pigs using the porcine single nucleotide polymorphism (SNP) 60 K beadchip and the PLINK program based on linear regression. Data used in the study included 410 F(2) pigs. All experimental animals were genotyped with 36,613 SNP markers located throughout the pig autosomes. We identified a strong association between a SNP marker on chromosome 7 and serum Ca(++) levels (DIAS0002191, genomic control-corrected P = 7.7 × 10(-5)). The position of DIAS0002191 was closely located to SLA class III region containing the C2 gene encoding the complementary component 2 protein, a protein which is important in the humoral immune responses. De novo sequencing of the porcine C2 gene revealed a missense mutation [c.1963A

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide.

Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Šresolution. The Ss-QAPRTase-NAMN cocrystals belonged to space group P321, with unit-cell parameters a=119.1, b=119.1, c=93.7 Å, γ=120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å3 Da(-1) and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.

Structural basis of E2-25K/UBB+1 interaction leading to proteasome inhibition and neurotoxicity.

E2-25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB(+1)) and has been identified as a crucial factor regulating amyloid-ß neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2-25K-UBB(+1) interaction, we determined the three-dimensional structures of UBB(+1), E2-25K and the E2-25K/ubiquitin, and E2-25K/UBB(+1) complex. The structures revealed that ubiquitin or UBB(+1) is bound to E2-25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2-25K mutants showed that disrupting UBB(+1) binding markedly diminishes synthesis of neurotoxic UBB(+1)-anchored polyubiquitin. These results suggest that the interaction between E2-25K and UBB(+1) is critical for the synthesis and accumulation of UBB(+1)-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.

Crystal structure of Helicobacter pylori MinE, a cell division topological specificity factor.

In Gram-negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure consists of an anti-MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long alpha-helix and two anti-parallel beta-strands, which mediate formation of a homodimeric alpha/beta structure. Here we report the crystal structure of full-length Helicobacter pylori MinE and redefine its TSD based on that structure. The N-terminal region of the TSD (residues 19-26), previously defined as part of the anti-MinCD domain, forms a beta-strand (betaA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti-parallel betaA-strands. Moreover, we observed serial dimer-dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti-parallel beta-strand interactions.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase.

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Šresolution. Native Hs-QPRTase crystals belonged to space group P2(1), with unit-cell parameters a=76.2, b=137.1, c=92.7 Å, ß=103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å3 Da(-1), which corresponds to a solvent content of 49.9%.

Characterization of calumenin-SERCA2 interaction in mouse cardiac sarcoplasmic reticulum.

Calumenin is a multiple EF-hand Ca(2+)-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265-269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca(2+)-cycling proteins. But it enhanced Ca(2+) transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca(2+) uptake showed increased Ca(2+) sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 mum Ca(2+), suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132-222 amino acids of calumenin and 853-892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca(2+)-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca(2+)-cycling of cardiomyocytes.

Differences in Muscle Fiber Characteristics and Meat Quality by Muscle Type and Age of Korean Native Black Goat.

To investigate the relationship between muscle fiber characteristics and meat quality traits by age of Korean native black goat (KNBG), four muscles (longissimus dorsi, LD; psoas major, PM; semimembranosus, SM; gluteus medius, GM) were obtained from five adult goat (AG; 18 months old) and five young goat (YG; 9 months old). PM muscle had the highest fiber number percentage (FNP) and fiber area percentage (FAP) of type I, followed by SM, GM, and LD muscles. FNP and FAP of type IIB were significantly (p<0.001) higher in AG than those in YG. YG had higher L* values but lower b* values than AG. The highest L* and b* values were observed in LD muscle (p<0.001). Age and muscle type had detrimental (p<0.001) effect on shear force and collagen content for all muscle in AG as compared to YG. YG had significantly (p<0.001) higher myofibrillar fragmentation index (MFI) than AG for all four muscles. These results suggest that muscle fiber compositions of different muscle types of KNBG depend on age, resulting in variations of meat color, MFI, collagen content, and shear force.

Effects of Intensive Alfalfa Feeding on Meat Quality and Fatty Acid Profile of Korean Native Black Goats.

The aim of this study was to determine meat quality characteristics and fatty acid composition of Korean native black goats (KNBG) finished on intensive feeding of alfalfa (ALF) and conventional feeding of commercial concentrate pellets (CCP) with low-energy common grasses. Ten KNBG (12 months old) were divided into two groups and subjected to either ALF or CCP treatments. The goats were slaughtered after 6 months of feeding with experimental diets to investigate meat quality characteristics and fatty acid compositions of longissimus lumborum muscle. There were no significant differences in proximate chemical composition, collagen, or myoglobin content between ALF and CCP groups of goats. Meat color, water-holding capacity, or tenderness was not significantly different between the two groups either. However, proportions of monounsaturated fatty acids and polyunsaturated fatty acids were significantly (p<0.05) different between the two groups. The proportion of oleic acid was significantly (p<0.05) higher in ALF goat whereas proportion of linoleic and arachidonic acids were significantly (p<0.05) higher in CCP goat. Results suggest that KNBG finished with intensive feeding of alfalfa could produce goat meat with desirable fatty acids for human diets.