Chipping at large, potent human T-cell leukemia virus type 1 protease inhibitors to uncover smaller, equipotent inhibitors

The human T-cell leukemia virus type 1 (HTLV-I) causes adult T-cell leukemia and several severe chronic diseases. HTLV-I protease (PR) inhibition stops the propagation of the virus. Herein, truncation studies were performed on potent octapeptidic HTLV-I PR inhibitor KNI-10161 to derive small hexapeptide KNI-10127 with some loss in activity. After performing residue-substitution studies on compound KNI-10127, HTLV-I PR inhibitory activity was recovered in inhibitor KNI-10166.     

Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V_max and K_m for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of “Candidatus Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.     

Preliminary X-ray diffraction analysis of a crystallizable mutant of malate dehydrogenase from the thermophile Thermus flavus

Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies. The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A. The asymmetric unit consists of one homodimer of molecular mass 67,000 Da. The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected.     

Efficient synthesis of (±)-parasitenone, a novel inhibitor of NF-κB

Dehydroxymethylepoxyquinomicin (DHMEQ, 1) is a novel nuclear factor-κB (NF-κB) inhibitor that inhibits DNA binding of NF-κB components including p65. To inspect its biological activity of 1, we synthesized parasitenone (3), possessing the common epoxycyclohexenone moiety of 1. Assessment of the inhibitory activity against NF-κB indicated that the epoxycyclohexenone moiety is the most essential element for the NF-κB inhibitory activity and the salicylic acid moiety may contribute the binding efficiency and specificity.     

Differences in the Regulatory and Functional Effects of the Us3 Protein Kinase Activities of Herpes Simplex Virus 1 and 2

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases with amino acid sequences that are conserved in the subfamily Alphaherpesvirinae (6, 24, 36). Based on studies showing that recombinant Us3 mutants of HSV-1 and HSV-2 have significantly impaired viral replication and virulence in mice models, it has been concluded that both HSV-1 and HSV-2 Us3 protein kinases play important roles in viral replication and pathogenicity in vivo (25, 33, 41). In contrast, HSV-1 and HSV-2 Us3 protein kinases are not essential for growth in tissue culture cells (33, 36). Thus, recombinant Us3 mutants grow as well as wild-type viruses in Vero cells, and the mutants exhibit modestly impaired replication in HEp-2 cells (33, 36, 39, 40). The possible functions of Us3 have been extensively studied and gradually elucidated for HSV-1 Us3, but much less is known about HSV-2 Us3. These functions include (i) blocking apoptosis (1, 22, 30, 31, 35); (ii) promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (39, 40, 45); (iii) redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (14, 37, 38) and cellular proteins, including lamin A/C and emerin (21, 27, 28); (iv) controlling infected cell morphology (13, 31, 32); and (v) downregulating cell surface expression of viral envelope glycoprotein B (gB) (12).To determine the molecular mechanisms for a viral protein kinase''s effects in infected cells, the kinase''s physiological substrates and its phosphorylation sites must be identified. This can involve studies showing that the altered phenotypes observed in cells infected with a mutant virus lacking the protein kinase activity is also detected in cells infected with a mutant virus in which the substrate''s phosphorylation sites have been modified by mutations. Although more than 15 potential HSV Us3 substrates have been reported, HSV-1 Us3 phosphorylation of only three substrates (Us3 itself, gB, and UL31) has been demonstrated to be linked directly with Us3 functions in infected cells (12, 13, 29, 41) as follows. (i) Us3 has been reported to autophosphorylate serine at position 147 (Ser-147), and this phosphorylation augments Us3''s kinase activity in infected cells (13, 41). Even though only a small fraction of Us3 is autophosphorylated at Ser-147 in infected cells, alanine replacement of Ser-147 in Us3 significantly reduced HSV-1 replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (41). These results indicated that Us3 kinase activity was, in part, regulated by autophosphorylation of Ser-147, and regulation of Us3 activity by autophosphorylation played a critical role in viral replication in vivo and HSV-1 pathogenesis. (ii) It has been reported that HSV-1 Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, and this phosphorylation downregulates the cell surface expression of gB (12). Us3 phosphorylation of gB at Thr-887 also has been proposed to be involved in the regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with mutant viruses carrying the amino acid substitution mutation T887A in gB and lacking the capacity to produce gH (45). The Us3 phosphorylation of gB at Thr-887 appeared to be critical for HSV-1 replication and pathogenesis in vivo, based on studies showing that the T887A substitution in the phosphorylation site in gB significantly reduced viral replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (Takahiko Imai, Ken Sagou, and Yasushi Kawaguchi, unpublished observations). (iii) It has been shown that Us3 phosphorylated some or all of the six serines in the UL31 N-terminal region, and this phosphorylation regulated the proper localization of UL31 and UL34 at the nuclear membrane and nuclear egress of nucleocapsids (29). Thus, the molecular basis of HSV-1 Us3 effects in infected cells have been gradually elucidated.However, the Us3 phosphorylation sites in Us3 itself and in gB are not conserved between HSV serotypes (12, 13). The amino acid residues in HSV-2 Us3 and gB corresponding to HSV-1 Us3 Ser-147 and gB Thr-887 are aspartic acid (Asp-147) and alanine (Ala-887), respectively. These results suggest that some HSV-1 Us3 functions, such as regulation of its own catalytic activity and control of gB expression on the cell surface, are not regulated by HSV-2 Us3 or are regulated in a manner(s) different from HSV-1 Us3. In agreement with this suggestion, there is a marked difference between HSV-1 and HSV-2 virulence in mice following intracerebral infection, with the HSV-1 Us3 null mutant being >10⁴-fold less virulent than the parent wild-type virus (25), while the HSV-2 Us3 null mutant was only ∼10-fold less virulent (33). Although these results were from different reports and the mouse strains used in the studies were different, they indicate that some HSV-1 Us3 functions are different from those of HSV-2 Us3.Therefore, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases. It was of particular interest to examine whether Asp-147 in HSV-2 Us3 is required for its own kinase activity, since it is well established that acidic amino acids such as Asp or glutamic acid sometimes mimic the negative charges produced by phosphorylation (29, 46). In the present study, using a genetic manipulation system of HSV-2 with our newly constructed HSV-2 bacterial artificial chromosome (BAC) clone, we have shown that HSV-2 Us3 exhibited marked differences from HSV-1 Us3 in its catalytic functions, including the regulation of UL31/UL34 localization, nuclear egress of nucleocapsids, cell surface expression of gB, and virulence in mice. We also found that Asp-147 in HSV-2 Us3 was critical for its kinase activity, raising a possibility that the activity of Us3 kinases was regulated differently in HSV-1 and HSV-2.     

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.