Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α

Purification and characterization of recombinant pyruvate dehydrogenase kinases from pea and soybean plants [abstract]

Abstract only availableFaculty Mentor: Dr. Douglas Randall, BiochemistryThe pyruvate dehydrogenase complex (PDC) is a large multienzyme complex catalyzing the oxidative decarboxylation of pyruvate and concomitant reduction of NAD to yield acetyl-CoA and NADH. The plant PDCs have vital roles in catabolic and anabolic metabolism. The plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Additionally, mitochondrial PDC (mtPDC) contains two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase. PDK catalyzes phosphorylation on the subunit of E1, resulting in inactivation of the complex. We have cloned two PDKs from soybean and recently we have cloned three PDKs from pea. cDNAs encoding soybean PDK 1 and 2 and pea PDK 1, 2 and 3 were subcloned into pET expression vector and E. coli BL21 (DE3) cells were transformed with each pET-28-H6-PDK construct. Recombinant proteins were expressed and purified by Ni-NTA agarose column chromatography to approximately 95% homogeneity. Biochemical characterization of these proteins is underway.The pyruvate dehydrogenase complex (PDC) is a large multienzyme complex catalyzing the oxidative decarboxylation of pyruvate and concomitant reduction of NAD to yield acetyl-CoA and NADH. The plant PDCs have vital roles in catabolic and anabolic metabolism. The plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Additionally, mitochondrial PDC (mtPDC) contains two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase. PDK catalyzes phosphorylation on the a subunit of E1, resulting in inactivation of the complex. We have cloned two PDKs from soybean and recently we have cloned three PDKs from pea. cDNAs encoding soybean PDK 1 and 2 and pea PDK 1, 2 and 3 were subcloned into pET expression vector and E. coli BL21 (DE3) cells were transformed with each pET-28-H6-PDK construct. Recombinant proteins were expressed and purified by Ni-NTA agarose column chromatography to approximately 95% homogeneity. Biochemical characterization of these proteins is underway

Subcellular localization of the Arabidopsis thaliana atDjC37 molecular chaperone protein

Abstract only availableThere are 94 genes encoding J-domain molecular chaperone proteins in the Arabidopsis thaliana genome. These genes have been grouped into 51 families (Miernyk 2001 Cell Stress Chaperones 6: 209-218). Family 4 consists of two proteins, atDjC6 and atDjC37. It has been previously determined that atDjC6 is nuclear localized (Suo & Miernyk 2004 Protoplasma 224: 79-89). We now wish to determine the subcellular localization of atDjC37. In silico analysis of the atDjC37 deduced amino acid sequence (http://maple.bioc.columbia.edu/predictNLS/) yielded the prediction that residues -R253RSSKKS- comprise a nuclear localization signal (NLS) sequence. Our experimental strategy has been to construct plasmids that encode full-length atDjC37 protein and a C-terminal truncated version that lacks the NLS sequence, fused to the red fluorescent protein. These proteins will be transiently expressed in biolistically-transformed tobacco BY2 cells, and localized using laser-scanning confocal microscopy. The transformed cells will be simultaneously incubated with a fluorescent nuclear stain to test for signal coincidence. Four nuclear stains are being evaluated for their utility; propidium iodide (PI), DAPI, SYTO Green, and Hoechst 33342. The SYTO and Hoechst 33342 stains are considered cell-permeant, while DAPI is "semi-permeant" and PI is impermeant. The PI and DAPI stains are UV blue-fluorescent, while PI is red and SYTO Green is, naturally, green.Plant Genomics Internship @ M

Analysis of family 24 of the Arabidopsis thaliana J-domain chaperone proteins [abstract]

Abstract only availableFaculty Mentor: Dr. Jan Miernyk, Plant BiochemistryThe Arabidopsis thaliana genome includes an unexpectedly large and diverse group of J-domain chaperone proteins. The 93 A. thaliana J-domain protein sequences have been grouped into 51 families, many of which do not have any well-studied counterpart in microbes or mammalian cells. Based upon the results of silico analyses, three proteins, atDjC43, atDjC48, and atDjC49, were assigned to Family 24. Homologous proteins are present in maize, rice, and soybean, and in a variety of animals, but none has been characterized. Members of Family 24 have approximately 300 amino acid residues; besides the J-domain the only prominent structural feature is a predicted transmembrane helix near the C-terminus. Preliminary experiments were conducted to try to understand the multiplicity of plant J-domain proteins, and to address the question of redundancy versus specialization. In order to determine where and when these three J-domain proteins are expressed, primers were designed for semi-quantitative RT-PCR. Total RNA was isolated from the rosette leaves and roots of 4-week old A. thaliana ecotype Columbia plants, and from flowers and green siliques. The results establish a pattern of organ-specific expression. We are also using T-DNA insertion knockout plants for our analyses. We currently have homozygous KO plants for atDjC48, and are in the final stages of screening for atDjC43 knockouts. It is difficult to express eukaryotic integral membrane proteins in bacteria. The sequence encoding the C-terminal transmembrane helix was deleted from the atDjC48 reading frame. The atDjC48���C sequence was then cloned into the pCal-n vector for expression in Escherichia coli as a chimera with the CaM-Binding-Peptide. The recombinant protein will be assayed for activity in vitro. Using these diverse strategies, we hope to gain insight into the roles of this Family of molecular chaperone proteins.University of Missouri--Columbia. Office of Undergraduate ResearchPlant Genomics Internship @ M

In silico analysis of protein Lys-Nε-acetylation in plants

Among posttranslational modifications, there are some conceptual similarities between Lys-NƐ-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. The study of Lys-acetylation of plant proteins has lagged behind studies of mammalian and microbial cells; thousands of acetylation sites have been identified in mammalian proteins compared with only hundreds of sites in plant proteins. While most previous emphasis was focused on posttranslational modifications of histones, more recent studies have addressed metabolic regulation. Being directly coupled with cellular CoA/acetyl-CoA and NAD/NADH, reversible Lys-NƐ-acetylation has the potential to control, or contribute to control, of primary metabolism, signaling, and growth and development

<it>In silico</it> biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide

Abstract Background Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure. Results The 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors. Conclusion We propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3’-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.</p

Do cupins have a function beyond being seed storage proteins? An updated working model for the growth and reproductive success of flax (Linum usitatissimum) in a radio-contaminated environment

Plants continue to flourish around the site of the Chernobyl Nuclear Power Plant disaster. The ability of plants to transcend the radio-contaminated environment was not anticipated and is not well understood. The aim of this study was to evaluate the proteome of flax (Linum usitatissimum L.) during seed filling by plants grown for a third generation near Chernobyl. For this purpose, seeds were harvested at 2, 4, and 6 weeks after flowering and at maturity, from plants grown in either non-radioactive or radio-contaminated experimental fields. Total proteins were extracted and the two-dimensional gel electrophoresis (2-DE) patterns analyzed. This approach established paired abundance profiles for 130 2-DE spots, e.g., profiles for the same spot across seed filling in non-radioactive and radio-contaminated experimental fields. Based on Analysis of Variance (ANOVA) followed by sequential Bonferroni correction, eight of the paired abundance profiles were discordant. Results from tandem mass spectrometry show that four 2-DE spots are discordant because they contain fragments of the cupin superfamily-proteins. Most of the fragments were derived from the N-terminal half of native cupins. Revisiting previously published data, it was found that cupin-fragments were also involved with discordance in paired abundance profiles of second generation flax seeds. Based on these observations we present an updated working model for the growth and reproductive success of flax in a radio-contaminated Chernobyl environment. This model suggests that the increased abundance of cupin fragments or isoforms and monomers contributes to the successful growth and reproduction of flax in a radio-contaminated environment

New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins