The retinoblastoma tumor suppressor protein (pRb) is a key negative regulator of cell proliferation that is frequently disregulated in human cancer. Many viral oncoproteins (for example, HPV E7 and E1A) are known to bind to the pRb pocket domain via a LXCXE binding motif. There are also some 20 cellular proteins that contain a LXCXE motif and have been reported to associate with the pocket domain of pRb. Using NMR spectroscopy and isothermal calorimetry titration, we show that LXCXE peptides of viral oncoproteins bind strongly to the pocket domain of pRb. Additionally, we show that LXCXE-like peptides of HDAC1 bind to the same site on pRb with a weak (micromolar) and transient association. Systematic substitution of residues other than conserved Leu, Cys, and Glu show that the residues flanking the LXCXE are important for the binding, whereas positively charged amino acids in the XLXCX-EXXX sequence significantly weaken the interaction.The retinoblastoma protein, pRb, is a 928-amino acid protein that belongs to the family of so-called pocket proteins (other members being p107 and p130) (1, 2). The small pRb pocket, which is the major focus of tumorigenic mutations in pRb, comprises the A and B cyclin-like domains (pRb-AB, amino acids 379 -578 and 641-791, respectively) (3-5). The tumor suppressor action of pRb stems from its ability to arrest cells at the G 1 phase of the cell cycle by suppressing the activity of the E2F family of transcription factors (6, 7). It is now believed that the pRb together with p53 tumor suppressor gene pathways is inactivated in most of the cancers (8).The small pocket of pRb binds to the LXCXE-like sequence-containing proteins (9) and includes also a primary binding site for E2Fs (10 -15). Biochemical and structural studies showed that the E2F peptide binding site is separated by ϳ30 Å from the LXCXE peptide binding site (16,17). There are other contact points on viral oncoproteins for pRb, but the major interaction is through the LXCXE motif. The crystal structure of the pRb-AB domain of SV40 large T antigen shows that two-thirds of the total surface interaction between the two proteins is via the LXCXE motif (18). This motif interacts with the pRb-AB on the B subdomain in an extended conformation exactly like the LXCXE peptide from HPV E7 (18,19). The large pocket region of pRb (amino acids 379 -928) is known to have additional binding to E2Fs and is necessary for binding to other cellular proteins, for example, MDM2 (20). About 120 proteins have been reported to physically interact with pRb, mostly through the pRb pocket domain (21,22).DNA tumor virus oncoproteins, such as adenovirus E1A, SV40 large T antigen, HPV E7, (23-26) and about 20 cellular proteins, such as HDAC1, 2 HDAC2, BRG1, cyclin D1, BRAC1, and plasminogen activator inhibitor (PAI), possess a LXCXE-like motif in their sequences (27)(28)(29)(30)(31)(32)(33)(34)(35)(36). The viral oncoproteins inactivate the pocket proteins by direct association through their LXCXE sequences. As viral oncoproteins utilize the LXCXE mo...

Mutants of Azospirillum brasilense Sp Cd, resistant to 5-fluorotryptophan (FT) excreted 3-indoleacetic acid (IAA), i.e., auxin, producing up to 16 μg/mL which was 30 times greater than the wild-type level. Under conditions of nitrogen fixation, the mutants excreted IAA up to 1 μg/mL, 10 times more than the wild type. However, none of the FT-resistant mutants of Azospirillum lipoferum Sp RG 20a excreted high levels of IAA. This was probably due to differences in the tryptophan and IAA biosynthetic steps between A. brasilense and A. lipoferum strains. Some of the FT-resistant mutants of A. brasilense Sp Cd showed a reduced feedback inhibition of anthranilate synthetase by tryptophan. The increased synthesis of tryptophan could explain the observed excretion of tryptophan and related metabolites. In addition, the IAA-overproducing mutants excreted other amino acids, probably owing to pleiotropic effects of deregulated tryptophan biosynthesis on amino acid metabolism. The growth patterns of some mutants excreting large amounts of IAA were almost identical to those of the wild type.

We describe an NMR method that directly monitors the influence of ligands on protein-protein interactions. For a two-protein interaction complex, the size of one component should be small enough (less than ca. 15 kDa) to provide a good quality (15)N((13)C) HSQC spectrum after (15)N((13)C) labeling. The size of the second unlabeled component should be large enough so that the molecular weight of the preformed complex is larger than ca. 40 kDa. When the smaller protein binds to a larger one, broadening of NMR resonances results in the disappearance of most of its cross-peaks in the HSQC spectrum. Addition of an antagonist that can dissociate the complex would restore the HSQC spectrum of the smaller component. The method directly shows whether an antagonist releases proteins in their wild-type folded states or whether it induces their denaturation, partial unfolding, or precipitation. We illustrate the method by studying lead compounds that have recently been reported to block the MDM2-p53 interaction. Activation of p53 in tumor cells by inhibiting its interaction with MDM2 offers new strategy for cancer therapy.

Out of 102 cases, 93 cases were histopathologically appendicitis, rest nine cases showed no evidence of inflammation so the rate of negative appendectomy was around 9%. On histopathology normal appendix was found in nine patients (8.9%), AA in 71 patients (69.6%), complicated appendicitis (CA) which includes perforated and gangrenous appendicitis was present in 22 patients (21.5%). Perforations were more common in patients who were younger than 5 years. >60% patients presented with CA when the duration of pain was >72 h. Presence of appendicolith increased the probability of CA.

Bromodomains represent an extensive family of evolutionarily conserved domains that are found in many chromatin-associated proteins such as histone acetyltransferases (HAT) and subunits of ATP-dependent chromatin-remodeling complexes. These domains are associated with acetylated lysine residues that bind both in vivo and in vitro; for example, they bind to the N-acetylated lysines of the histone tail of nucleosomes. In this report, we determined the structure of the bromodomain from human brahma-related gene 1 (BRG1) protein, a subunit of an ATP-dependent switching/sucrose nonfermenting (SWI/SNF) remodeling complex, and have also characterized its in vitro interaction with N-acetylated lysine peptides from histones. In addition to a typical all-alpha-helical fold that was observed in the bromodomains, we observed for the first time a small beta-sheet in the ZA loop region of the BRG1 protein. The BRG1 bromodomain exhibited binding, albeit weak, to acetylated peptides that were derived from histones H3 and H4. We have compared the acetyl-lysine binding sites of BRG1 bromodomain with the yGCN5 (general control of amino acid biosynthesis). By modeling the acetylated-lysine peptide into the BRG1 bromodomain structure, we were able to explain the weak binding of acetylated-lysine peptides to this bromodomain.

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC1.1.3.4 ]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. RecombinantEscherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml−1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg−1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX inE. coli.

H/ACA ribonucleoprotein particles are essential for ribosomal RNA and telomerase RNA processing and metabolism. Shq1p has been identified as an essential eukaryotic H/ACA small nucleolar (sno) ribonucleoparticle (snoRNP) biogenesis and assembly factor. Shq1p is postulated to be involved in the early biogenesis steps of H/ACA snoRNP complexes, and Shq1p depletion leads to a specific decrease in H/ACA small nucleolar RNA levels and to defects in ribosomal RNA processing. Shq1p contains two predicted domains as follows: an N-terminal CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, and a C-terminal region of high sequence homology called the Shq1 domain. Here we report the crystal structure and functional studies of the Saccharomyces cerevisiae Shq1p CS domain. The structure consists of a compact antiparallel ␤-sandwich fold that is composed of two ␤-sheets containing four and three ␤-strands, respectively, and a short ␣-helix. Deletion studies showed that the CS domain is required for the essential functions of Shq1p. Point mutations in residues Phe-6, Gln-10, and Lys-80 destabilize Shq1p in vivo and induce a temperature-sensitive phenotype with depletion of H/ACA small nucleolar RNAs and defects in rRNA processing. Although CS domains are frequently found in co-chaperones of the Hsp90 molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA snoRNP biogenesis in vivo, possibly in an Hsp90-independent manner.Modification of uridine to pseudouridine in ribosomal RNA and some spliceosomal RNAs is catalyzed by highly specialized ribonucleoparticle (RNP) 3 complexes called box H/ACA RNPs (1-5). Depending on their site of maturation and action H/ACA RNPs are classified into two classes, small nucleolar RNPs (snoRNPs) and small Cajal body RNPs. In Saccharomyces cerevisiae, H/ACA snoRNPs contain four proteins: Nhp2p (L7ae in archaea (6) and Cbf5p, also called dyskerin, in humans (7)), Nop10p, Gar1p, and a single small nucleolar RNA (snoRNA), specific to each snoRNP (8 -11). Cbf5p provides the pseudouridylase activity to the complex, and the snoRNA component provides the "guide RNA" for positioning the substrate RNA for modification (8,10,(12)(13)(14)(15). The 3Ј end of human telomerase RNA (hTR) contains an H/ACA scaRNA domain that binds the H/ACA proteins and is required for 3Ј end processing, accumulation, and localization of hTR to Cajal bodies (16 -19). In archaea, the assembly of H/ACA snoRNP appears to proceed by assembly of the protein components, followed by the incorporation of the H/ACA RNA (8, 20 -23). In eukaryotes, the assembly and final maturation of the holoenzyme RNP are more complicated, possibly because of subcellular compartmentalization, and require accessory proteins (22, 24). Two proteins specifically found in eukaryotes, Naf1p and Shq1p, were initially identified in yeast as factors involved in the assembly of H/ACA snoRNPs (23-25). Both Shq1p and Naf1...

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