Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana.

Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. As an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolite-gene associations led to the identification of N-malonyl-D-allo-isoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5'-phosphate for its activity. This study thus identifies a new d-amino acid racemase gene family and advances our knowledge of plant d-amino acid metabolism that is currently largely unexplored. It also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism.

Emerging knowledge of regulatory roles of D-amino acids in bacteria.

The D-enantiomers of amino acids have been thought to have relatively minor functions in biological processes. While L-amino acids clearly predominate in nature, D-amino acids are sometimes found in proteins that are not synthesized by ribosomes, and D-Ala and D-Glu are routinely found in the peptidoglycan cell wall of bacteria. Here, we review recent findings showing that D-amino acids have previously unappreciated regulatory roles in the bacterial kingdom. Many diverse bacterial phyla synthesize and release D-amino acids, including D-Met and D-Leu, which were not previously known to be made. These noncanonical D-amino acids regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities. Elucidating the mechanisms by which D-amino acids govern cell wall remodeling and biofilm disassembly will undoubtedly reveal new paradigms for understanding how extracytoplasmic processes are regulated as well as lead to development of novel therapeutics.

Alzheimer's disease and amyloid beta-peptide deposition in the brain: a matter of 'aging'?

Biomolecules can experience aging processes that limit their long-term functionality in organisms. Typical markers of protein aging are spontaneous chemical modifications, such as AAR (amino acid racemization) and AAI (amino acid isomerization), mainly involving aspartate and asparagine residues. Since these modifications may affect folding and turnover, they reduce protein functionality over time and may be linked to pathological conditions. The present mini-review describes evidence of AAR and AAI involvement in the misfolding and brain accumulation of Abeta (amyloid beta-peptide), a central event in AD (Alzheimer's disease) synaptic dysfunctions. Structural alterations introduced by site-specific modifications linked to protein aging may affect Abeta production, polymerization and clearance, and therefore play a pivotal role in the pathogenesis of sporadic and genetic forms of AD. Early changes associated with molecular aging also have significant long-term consequences for Abeta folding and turnover. New fast, reproducible and accurate methods for the screening of protein aging markers in biological samples may contribute to improve diagnostic and therapeutic approaches in AD.

Proline cis-trans isomerization controls autoinhibition of a signaling protein.

Autoinhibition is being widely used in nature to repress otherwise constitutive protein activities and is typically regulated by extrinsic factors. Here we show that autoinhibition can be controlled by an intrinsic intramolecular switch afforded by prolyl cis-trans isomerization. We find that a proline on the linker tethering the two SH3 domains of the Crk adaptor protein interconverts between the cis and trans conformation. In the cis conformation, the two SH3 domains interact intramolecularly, thereby forming the basis of an autoinhibitory mechanism. Conversely, in the trans conformation Crk exists in an extended, uninhibited conformation that is marginally populated but serves to activate the protein upon ligand binding. Interconversion between the cis and trans, and, hence, of the autoinhibited and activated conformations, is accelerated by the action of peptidyl-prolyl isomerases. Proline isomerization appears to make an ideal switch that can regulate the kinetics of activation, thereby modulating the dynamics of signal response.

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity.

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation.

Nucleotides modulate the activity of aspartate racemase of Scapharca broughtonii.

The activity of D-aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in V(max) and not in K(m). Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and D-aspartate in energy metabolism under anoxic conditions are discussed.

Glutamate racemase is an endogenous DNA gyrase inhibitor.

Almost all bacteria possess glutamate racemase to synthesize d-glutamate as an essential component of peptidoglycans in the cell walls. The enforced production of glutamate racemase, however, resulted in suppression of cell proliferation. In the Escherichia coli JM109/pGR3 clone, the overproducer of glutamate racemase, the copy number (i.e. replication efficiency) of plasmid DNA declined dramatically, whereas the E. coli WM335 mutant that is defective in the gene of glutamate racemase showed little genetic competency. The comparatively low and high activities for DNA supercoiling were contained in the E. coli JM109/pGR3 and WM335 cells, respectively. Furthermore, we found that the DNA gyrase of E. coli was modulated by the glutamate racemase of E. coli in the presence of UDP-N-acetylmuramyl-l-alanine, which is a peptidoglycan precursor and functions as an absolute activator for the racemase. This is the first finding of the enzyme protein participating in both d-amino acid metabolism and DNA processing.

Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12.

FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems. Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial. FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine receptor (RyR1). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6. To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium. About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.

Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA.

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Functional analysis of the yeast 40 kDa cyclophilin Cyp40 and its role for viability and steroid receptor regulation.

We have identified and characterized a homolog of the 40 kDa cyclophilins in the budding yeast Saccharomyces cerevisiae. At the amino acid level, this novel yeast cyclophilin, termed Cyp40, is 47% identical to human cyclophilin-40. Recombinant Cyp40 produced in bacteria has a peptidyl-prolyl cis-trans isomerase activity with a catalytic efficiency (k[cat]/K[m]) of 0.5 x 10(6)M(-1)s(-1), which can be inhibited by cyclosporin A with an IC50 value of 60nM. Using a polyclonal antibody against Cyp40 we have found that Cyp40 is predominantly cytoplasmic, and that its expression is induced 3-4-fold by heat shock. Moreover, Cyp40 can be coprecipitated from yeast extracts with the cytosolic molecular chaperone Hsp90. Surprisingly, a Cyp40-deficient yeast strain is fully viable at normal and elevated temperatures. Cyp40 is also dispensable for normal regulation of vertebrate steroid receptors in yeast. While other immunophilins could conceivably compensate a Cyp40 defect, our results are compatible with the notion that immunophilins may be fortuitous partners in the biochemically established steroid receptor-Hsp90 complex.

Host-cell cyclophilin is important for the intracellular replication of Leishmania major.

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host-parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.

Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding.

The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. In its enzymatic mechanism the trigger factor follows the Michaelis-Menten equation. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 microM. In contrast, the catalytic constant kcat is small and shows a value of 1.3 s(-1) at 15 degrees C. An unfolded protein inhibits the trigger factor in a competitive fashion. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. Together, this suggests that the good substrate binding, i.e. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding.

A cyclophilin function in Hsp90-dependent signal transduction.

Cpr6 and Cpr7, the Saccharomyces cerevisiae homologs of cyclophilin-40 (CyP-40), were shown to form complexes with Hsp90, a protein chaperone that functions in several signal transduction pathways. Deletion of CPR7 caused severe growth defects when combined with mutations that decrease the amount of Hsp90 or Sti1, another component of the Hsp90 chaperone machinery. The activities of two heterologous Hsp90-dependent signal transducers expressed in yeast, glucocorticoid receptor and pp60(v-src) kinase, were adversely affected by cpr7 null mutations. These results suggest that CyP-40 cyclophilins play a general role in Hsp90-dependent signal transduction pathways under normal growth conditions.

Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.

Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple chaperone components that maintain substrates in an intermediate folded state.