Characterization of two β-decarboxylating dehydrogenases from <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg²⁺, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.     

Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39

One of the major pathological hallmarks of Alzheimer disease is neurofibrillary tangles. Neurofibrillary tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is one of the tau protein kinases that increase paired helical filament epitopes in tau by phosphorylation. Recently, various mutations of tau have been identified in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we investigated the phosphorylation of FTDP-17 mutant tau proteins, K257T, P301L, P301S, and R406W, by Cdk5 complexed with p35, p25, or p39 in vitro and in cultured cells. The extent of phosphorylation by all Cdk5 species was slightly lower in mutant tau than in wild-type tau. Major phosphorylation sites, including Ser202, Ser235, and Ser404, were the same among the wild-type, K257T, P301L, and P301S tau proteins phosphorylated by any Cdk5. On the other hand, R406W tau was less phosphorylated at Ser404 than were the other variants. This was not due to the simple replacement of amino acid Arg406 with Trp close to the phosphorylation site, because Ser404 in a R406W peptide was equally phosphorylated in a wild-type peptide. The decreased phosphorylation of mutant tau by Cdk5s was canceled when tau protein bound to microtubules was phosphorylated. These results indicate that FTDP-17 mutations do not affect the phosphorylatability of tau by Cdk5 complexed with p35, p25, or p39 and may explain part of the discrepancy reported previously between in vivo and in vitro phosphorylation of FTDP-17 tau mutants.     

The proteasome activator PA28 functions in collaboration with Hsp90 in vivo

We have previously shown that the proteasome activator PA28 is essential to Hsp90-dependent protein refolding in vitro, where PA28 mediates transfer of the Hsp90-bound substrate protein to the Hsc70/Hsp40 chaperone machine for its correct refolding. This observation suggests that PA28 may also collaborate with Hsp90 in cells. To examine this possibility, here we have used double-stranded RNA interference (RNAi) against PA28 in Caenorhabditis elegans mutants of daf-21, which encodes Hsp90. We show that C. elegans PA28 facilitates Hsp90-initiated protein refolding, albeit with an activity lower than that of mouse PA28 proteins. RNAi-mediated knockdown of PA28 significantly suppresses the Daf-c (dauer formation constitutive) phenotype of the daf-21 mutant, but it has no affect on the distinct defects of this mutant in sensing odorants. Taking these results together, we conclude that PA28 is likely to function in collaboration with Hsp90 in vivo.     

Metabolome analysis revealed increase in S-methylcysteine and phosphatidylisopropanolamine synthesis upon L-cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica

L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.     

Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL^pro. A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL^pro motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1′ site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC₅₀ or K_i values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K_i values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL^pro may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1′-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.     

Anti-Inflammatory Effects of Rebamipide Eyedrop Administration on Ocular Lesions in a Murine Model of Primary Sj?gren's Syndrome

Background

Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb), a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren''s syndrome (SS). However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown.

Methods and Finding

Reb eyedrops were administered to the murine model of SS aged 4–8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment.

Conclusion

Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces.     

Rax1, a protein required for the establishment of the bipolar budding pattern in yeast

In Saccharomyces cerevisiae, cell type determines two distinct spatial budding patterns. Haploid cells exhibit an axial pattern, whereas diploid cells exhibit a bipolar pattern. Axl1, a member of the insulin-degrading enzyme (IDE) family, is the key morphological determinant for the haploid axial pattern. Here we identified a novel gene, RAX1, specifically required for the bipolar budding pattern. Loss of RAX1 alters the bipolar pattern of axl1 haploids resulting in reversion to the axial pattern, and also alters the bipolar patterns of bud3 and bud4 haploids. However, bud10 rax1 haploids exhibit a random budding pattern, suggesting Bud10 acts as the key proximal landmark in axial budding. Rax1 is required for the localization of Bud8, the distal bipolar budding landmark. Interestingly, Rax1 contains a C-terminal domain possessing some similarity to insulin-related peptides. Our results suggest that Rax1 is necessary for the establishment of the bipolar budding landmark.