Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to stimulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20-25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same Km for the substrate 4-coumaroyl-CoA but a lower Vmax than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.

The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner.

A cDNA clone for a cysteine proteinase inhibitor of rice (oryzacystatin) was isolated from a lambda gt10 cDNA library of rice immature seeds by screening with synthesized oligonucleotide probes based on partial amino acid sequences of oryzacystatin. A nearly full-length cDNA clone was obtained which encoded 102-amino acid residues. The amino acid sequence of oryzacystatin deduced from the cDNA sequence was significantly homologous to those of mammalian cystatins, especially family 2 cystatins. Oryzacystatin contained the sequence Gln-Val-Val-Ala-Gly conserved among most members of the cystatin superfamily. The gene for oryzacystatin was transcribed into a single mRNA species of about 700 nucleotides. The content of mRNA reached its highest level 2 weeks after flowering and then gradually decreased to undetectable levels at 10 weeks. This feature of transient expression is coordinate with that of glutelin (a major storage protein), although the expression of oryzacystatin precedes that of glutelin by about 1 week.

A highly conserved position of negatively charged amino acids is present in the SS2 segments of the S5-S6 linker regions among calcium channels. We report here that replacing Glu residues at this position alters the ion selectivity of the human cardiac calcium channel. Substituting Glu334 in motif I or Glu1086 in motif III with Lys produced mutant calcium channels that permeated sodium ions 10-fold more effectively than barium ions. More conservative changes such as substitution of Glu1086 with Gln or substitution of Glu1387 with Ala also increased sodium permeation through the mutant calcium channels. Sodium currents through the mutant calcium channels could be modulated by dihydropyridines and blocked by external divalent cations. These results suggest that Glu334, Glu1086, and Glu1387 are part of a ring of glutamate residues formed in the pore-lining SS1-SS2 region and are critical in determining ion selectively and permeability of a human cardiac calcium channel.

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 and CTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1 is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1(up2), is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to the MAC1(up1) allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA ... TGAGCAAA, appearing within the 5'-flanking region of the CTR1 promoter, is necessary and sufficient for the copper- and MAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in the FRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264-279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

The role of lysines in the ribonucleolytic and angiogenic activities of human angiogenin has been examined by chemical modification and site-directed mutagenesis. It was demonstrated previously [Shapiro, R., Weremowicz, S., Riordan, J.F., & Vallee, B.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787] that extensive treatment with lysine reagents markedly decreases the ribonucleolytic activity of angiogenin. In the present study, limited chemical modification with 1-fluoro-2,4-dinitrobenzene followed by C18 high-performance liquid chromatography yielded several (dinitrophenyl)angiogenin derivaties. The major derivative formed had slightly increased enzymatic activity compared with the unmodified protein. Tryptic peptide mapping demonstrated the site of modification to be Lys-50. A second derivative, modified at Lys-60, was 34% active. Analysis of a third derivative indicated that modification of Lys-82 did not decrease activity. Thus, Lys-50 and Lys-82 are unessential for enzymatic activity while Lys-60 may play a minor role. No pure derivative modified at Lys-40, corresponding to the active-site residue Lys-41 of the homologous protein ribonuclease A, could be obtained by chemical procedures. Therefore, we employed oligonucleotide-directed mutagenesis to replace this lysine with glutamine or arginine. The Gln-40 derivative had less than 0.05% enzymatic activity compared with the unmodified protein and substantially reduced angiogenic activity when examined with the chick embryo chorioallantoic membrane assay. These results suggest that the angiogenic activity of the protein is dependent on an intact enzymatic active site. The Arg-40 derivative had 2.2% ribonucleolytic activity compared with unmodified angiogenin. The effects of reductive methylation of this derivative indicate that no lysines other than Lys-40 are critical.(ABSTRACT TRUNCATED AT 250 WORDS)

Patients under age 40 constitute 35.6% of all colorectal cancer cases in Egypt, an unusual disease pattern to which both environmental exposures and inefficient DNA repair may contribute. While a number of polymorphisms in DNA repair genes have been recently identified, their role as cancer risk modifiers is yet to be determined. In a pilot case-control study, we tested the hypothesis that polymorphisms in the gene for the DNA repair enzyme XRCC1 are associated with increased risk of colorectal cancer among Egyptians. Using a multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology, allelic variants of the XRCC1 gene at codons 194 (Arg->>Trp) (194Trp) and 399 (Arg->>Gln) (399Gln), were analyzed in DNA from lymphocytes of 48 newly-diagnosed colorectal cancer cases and 48 age- and sex-matched controls. Overall, the inheritance of 194Trp allele (Arg/Trp genotype) and 399Gln allele (combined Arg/Gln and Gln/Gln genotypes) was associated with increased colorectal cancer risk (odds ratio (OR)=2.56, 95% confidence limits (CL) 0.73-9.40, and P=0. 08 for 194Trp allele and OR=3.98, 95% CL 1.50-10.6, and P<0.001 for 399Gln allele). Interestingly, the frequencies of 194Trp and 399Gln genotypes were higher in colorectal cancer cases under age 40 than in corresponding controls, and an association between both polymorphisms and early age of disease onset was observed (OR=3.33, 95% CL 0.48-35.90, and P=0.16 for 194Trp and OR=11.90, 95% CL 2.30-51.50, and P=0.0003 for 399Gln). Analysis of the data after adjustment for place of residence indicated that the frequencies of the genotypes with the 194Trp and the 399Gln alleles were higher among urban residents (OR=3.33, 95% CL 0.48-35.90, and P=0.16 for 194Trp and OR=9.97, 95% CL 1.98-43.76, and P<0.001 for 399Gln) than among rural residents (OR=2.00, 95% CL 0.36-26.00, and P=0.30 for 194Trp and OR=1.90, 95% CL 0.50-7.53, and P=0.20 for 399Gln). These findings support our hypothesis and suggest that polymorphisms in the XRCC1 gene, in conjunction with place of residence, may modify disease risk. This first demonstration that polymorphisms in DNA repair genes may contribute to colorectal cancer susceptibility and may increase the risk of early onset of the disease opens the door for future studies in that direction.

Post-translational modifications of the water-soluble human lens crystallins from young adult donors were identified and located using electrospray ionization mass spectrometric analysis of the intact proteins and fast atom bombardment mass spectrometry of enzymatic digests. Peptides corresponding to all of the sequences of alpha A-, alpha B-, and beta B2-crystallins were found, permitting the entire sequences to be searched for modifications. The major portions of these three crystallins were not modified. Modifications of alpha A-crystallin that were detected included 2 phosphorylated Ser residues (1 of which appears to be unique to human lenses), deamidation at some Gln and Asn residues, a disulfide bond between Cys-131 and Cys-142, and loss of the COOH-terminal Ser residue. Three phosphorylated Ser residues, but no deamidation, were found in alpha B-crystallin. The molecular weights of neither the intact protein nor the peptides in the enzymatic digests indicated any post-translational modification of the principal beta-crystallin, beta B2. The molecular weights of the other beta- and gamma-crystallins for which sequences have been published suggested the presence of post-translational modifications or errors in the published sequences. Although enough peptides were found to establish the presence of specific proteins, peptides corresponding to all portions of these proteins were not found, and elucidation of these structures is not yet complete. This mass spectrometric characterization of the total water-soluble proteins from normal young adult lenses provides a reference data base for future investigations of the modifications present in aged and cataractous lenses.

Human monoclonal antibodies 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen-binding structural elements including an elongated CDR H3 forming main-chain interactions with the N terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-pi sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position.

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu(1) conversion is favored at pH 6.0 while Gln(1) conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.

Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA(Gln) into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA(CUG)(Gln) and tRNA(UUG)(Gln) species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA(Gln) import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA(Gln) species and a bacterial pre-tRNA(Asp) can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. A nine-amino acid peptide derived from ADNF (Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; ADNF-9) captured the activity of the parent protein and has been reported to protect cultured neurons from multiple neurotoxins. Antibodies recognizing ADNF-9 produced neuronal apoptosis, and identified an additional, structurally related, glia-derived peptide, Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (NAP). Previous comparative studies have characterized s.c.-injected NAP as most efficacious in protecting against developmental retardation and learning impairments in apolipoprotein E-deficient mice. This study was designed to assess 1) neuroprotection after intranasal administration of ADNF-9 and NAP to rats treated with the cholinotoxin ethylcholine aziridium; and 2) bioavailability and pharmacokinetics after intranasal administration. Results showed significant improvements in short-term spatial memory, as assessed in a water maze, after daily intranasal administration of 1 microg of peptide (ADNF-9 or NAP) per animal. However, a 5-day pretreatment with ADNF-9 did not improve performance measured after cessation of treatment. Compared with rats treated with ADNF-9, NAP-pretreated animals exhibited a significantly better performance. Furthermore, NAP (and not ADNF-9) protected against loss of choline acetyl transferase activity. Significant amounts of (3)H-labeled NAP reached the brain, remained intact 30 min after administration, and dissipated 60 min after administration. This study revealed efficacy for ADNF-related peptides in rodent models for neurodegeneration. The small size of the molecules, the low dosage required, the noninvasive administration route, and the demonstrated activity in a relevant paradigm suggest NAP as a lead compound for future drug design.

Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.

The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 ->> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.

To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.

BACKGROUND

The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East.

RESULTS

This scheme uses 7 loci; atpD, fusA, glnS, gltB, gyrB, infB, and pps. There were 12 sequence types (ST) identified in C. sakazakii, and 3 in C. malonaticus. A third (22/60) of C. sakazakii strains were in ST4, which had almost equal numbers of clinical and infant formula isolates from 1951 to 2008. ST8 may represent a particularly virulent grouping of C. sakazakii as 7/8 strains were clinical in origin which had been isolated between 1977 - 2006, from four countries. C. malonaticus divided into three STs. The previous Cronobacter biotyping scheme did not clearly correspond with STs nor with species.

CONCLUSIONS

In conclusion, MLST is a more robust means of identifying and discriminating between C. sakazakii and C. malonaticus than biotyping. The MLST database for these organisms is available online at http://pubmlst.org/cronobacter/.

Resistance to activated protein C (APC) is the most prevalent inherited cause of venous thrombosis. The APC resistance phenotype is associated with a single point mutation in the factor V gene, changing Arg506 in the APC cleavage site to a Gln. We have investigated 50 Swedish families with inherited APC resistance for this mutation and found it to be present in 47 of them. Perfect cosegregation between a low APC ratio and the presence of mutation was seen in 40 families. In seven families, the co-segregation was not perfect as 12 out of 57 APC-resistant family members were found to lack the mutation. Moreover, in three families with APC resistance, the factor V gene mutation was not found, suggesting another still unidentified cause of inherited APC resistance. Of 308 investigated families members, 146 were normal, 144 heterozygotes, and 18 homozygotes for the factor V gene mutation and there were significant differences in thrombosis-free survival curves between these groups. By age 33 yr, 8% of normals, 20% of heterozygotes, and 40% of homozygotes had had manifestation of venous thrombosis.

Hydrogen rearrangement is an important process in radical chemistry. A high degree of H. rearrangement to and from z. ionic fragments (combined occurrence frequency 47% compared with that of z.) is confirmed in analysis of 15,000 tandem mass spectra of tryptic peptides obtained with electron capture dissociation (ECD), including previously unreported double H. losses. Consistent with the radical character of H. abstraction, the residue determining the formation rate of z' = z. + H. species is found to be the N-terminal residue in z. species. The size of the complementary c(m)' fragment turned out to be another important factor, with z' species dominating over z. ions for m < or = 6. The H. atom was found to be abstracted from the side chains as well as from alpha-carbon groups of residues composing the c' species, with Gln and His in the c' fragment promoting H. donation and Asp and Ala opposing it. Ab initio calculations of formation energies of .A radicals (A is an amino acid) confirmed that the main driving force for H. abstraction by z. is the process exothermicity. No valid correlation was found between the NC(alpha) bond strength and the frequency of this bond cleavage, indicating that other factors than thermochemistry are responsible for directing the site of ECD cleavage. Understanding hydrogen attachment to and loss from ECD fragments should facilitate automatic interpretation ECD mass spectra in protein identification and characterization, including de novo sequencing.

Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the pathological expansion of a glutamine tract. A hallmark of these so-called polyglutamine diseases is the presence of ubiquitylated inclusion bodies, which sequester various components of the 19S and 20S proteasomes. In addition, the ubiquitin-proteasome system (UPS) has been shown to be severely impaired in vitro in cells overexpressing mutant huntingtin. Thus, because of its fundamental housekeeping function, impairment of the UPS in neurons could contribute to neurotoxicity. We have recently proposed that the proteasome activator REGgamma could contribute to UPS impairment in polyglutamine diseases by suppressing the proteasomal catalytic sites responsible for cleaving Gln-Gln bonds. Capping of proteasomes with REGgamma could therefore contribute to a potential 'clogging' of the proteasome by pathogenic polyglutamines. We show here that genetic reduction of REGgamma has no effect on the well-defined neurological phenotype of R6/2 HD mice and does not affect inclusion body formation in the R6/2 brain. Surprisingly, we observe increased proteasomal 'chymotrypsin-like' activity in 13-week-old R6/2 brains relative to non-R6/2, irrespective of REGgamma levels. However, assays of 26S proteasome activity in mouse brain extracts reveal no difference in proteolytic activity regardless of R6/2 or REGgamma genotype. These findings suggest that REGgamma is not a viable therapeutic target in polyglutamine disease and that overall proteasome function is not impaired by trapped mutant polyglutamine in R6/2 mice.

We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb-YQR [Leu-(B10)->>Tyr; His(E7)->>Gln; Thr(E10)->>Arg] to 1.4-A resolution by ultra-low temperature (20 K) x-ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe-CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 A from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849-2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb-YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild-type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701-705] and room temperature [Srajer et al. (1996) Science 274, 1726-1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein.

The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.

We have identified cDNA clones coding for the major sulphur-rich and sulphur-poor groups of barley storage proteins (the B- and C-hordeins, respectively). Hybridization studies have revealed unexpected homologies between B- and C-hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C-hordein-related sequences within, or closely associated with, B-hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B-hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline-rich, sulphur-poor), which is homologous to C-hordein sequences, and domain 2 (non-repetitive, proline-poor, sulphur rich), which makes up two-thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B- and C-hordein mRNAs have an asymmetric base composition (>80% C-A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. We discuss the evolutionary implications of the domain structure of the B-hordeins and the unusual relationship between the two groups of barley storage proteins.

A total of 399 independent mutants of Escherichia coli were obtained which have point and insertion mutations in the glnA region. Mutants isolated included Gln- and Reg- strains (unable to utilize arginine as a nitrogen source). Mutations were mapped with 73 deletion-containing derivatives of a lambda gln phage. Complementation analysis was performed with lambda gln derivatives containing point mutations which conferred a Gln- or Reg- phenotype. Deletion mapping and complementation analysis assigned 104 mutations in 24 deletion intervals to glnA. Mutations in Reg- strains were assigned to two genes, glnL and glnG. glnL contained 131 mutations in 12 deletion intervals, and glnG contained 164 mutations in 10 deletion intervals. The gene order is glnA-glnL-glnG, transcribed from left to right. Polarity of insertion mutations indicates that glnL and glnG form from left to right. Polarity of insertion mutations indicates that glnL and glnG form an operon. Complementation analysis of glnA insertion mutations with glnL and glnG mutations showed polarity of glnA onto most glnL and glnG alleles, suggesting that transcription of glnA may proceed into the glnL-glnG operon. All mutations analyzed in glnA conferred a Gln- phenotype. However, we also found that over half of the Gln- strains isolated ater chemical mutagenesis contained point mutations in glnG. Mutants which synthesized a high level of glutamine synthetase in the presence of ammonia (GlnC phenotype) were selected as revertants of a strain with a Tn10 insertion in glnD and were mapped with chromosomal deletions. Results indicate that mutations in 12 and 15 examined strains clearly map outside of glnA, probably in glnL.

The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.