Pyruvate dehydrogenase complex (PDC) is a large multienzyme complex that catalyzes the irreversible conversion of pyruvate to acetyl-coenzyme A with reduction of NAD+. Distinctive from PDCs in lower ...forms of life, in mammalian PDC, dihydrolipoyl acetyltransferase (E2; E2p in PDC) and dihydrolipoamide dehydrogenase binding protein (E3BP) combine to form a complex that plays a central role in the organization, regulation, and integration of catalytic reactions of PDC. However, the atomic structure and organization of the mammalian E2p/E3BP heterocomplex are unknown. Here, we report the structure of the recombinant dodecahedral core formed by the C-terminal inner-core/catalytic (IC) domain of human E2p determined at 3.1 Å resolution by cryo electron microscopy (cryoEM). The structure of the N-terminal fragment and four other surface areas of the human E2p IC domain exhibit significant differences from those of the other E2 crystal structures, which may have implications for the integration of E3BP in mammals. This structure also allowed us to obtain a homology model for the highly homologous IC domain of E3BP. Analysis of the interactions of human E2p or E3BP with their adjacent IC domains in the dodecahedron provides new insights into the organization of the E2p/E3BP heterocomplex and suggests a potential contribution by E3BP to catalysis in mammalian PDC.
FFQ, food diaries and 24 h recall methods represent the most commonly used dietary assessment tools in human studies on nutrition and health, but food intake biomarkers are assumed to provide a more ...objective reflection of intake. Unfortunately, very few of these biomarkers are sufficiently validated. This review provides an overview of food intake biomarker research and highlights present research efforts of the Joint Programming Initiative 'A Healthy Diet for a Healthy Life' (JPI-HDHL) Food Biomarkers Alliance (FoodBAll). In order to identify novel food intake biomarkers, the focus is on new food metabolomics techniques that allow the quantification of up to thousands of metabolites simultaneously, which may be applied in intervention and observational studies. As biomarkers are often influenced by various other factors than the food under investigation, FoodBAll developed a food intake biomarker quality and validity score aiming to assist the systematic evaluation of novel biomarkers. Moreover, to evaluate the applicability of nutritional biomarkers, studies are presently also focusing on associations between food intake biomarkers and diet-related disease risk. In order to be successful in these metabolomics studies, knowledge about available electronic metabolomics resources is necessary and further developments of these resources are essential. Ultimately, present efforts in this research area aim to advance quality control of traditional dietary assessment methods, advance compliance evaluation in nutritional intervention studies, and increase the significance of observational studies by investigating associations between nutrition and health.
Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH ...phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space group P3221, with unit‐cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular‐replacement methods and refined to a final R factor of 21.9% (Rfree = 24.7%). The final model consists of 402 of a possible 467 amino‐acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.
The crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase provides new insights into the mechanism of the regulation of the activity of the pyruvate dehydrogenase complex.
Premature migration is a key component of the biocomplexity of anadromous fishes, yet remains poorly understood. Many Atlantic salmon (Salmo salar) populations leave the ocean in spring, months prior ...to spawning, though this curtails feeding in productive marine environments. We hypothesized that habitat features encourage the evolution of this trait by providing fish with physical and thermal refuge during their long freshwater holding period. We document substantial variation in Atlantic salmon return-migration timing across 70 Irish rivers using 8 years of angling data, validated with electronic-counter data from 23 of the rivers. A higher frequency of spring migrating salmon was observed in rivers with accessible lakes and larger rivers in general. Spring migration may have evolved in rivers with suitable holding habitat as a strategy to minimize cumulative marine mortality, which is traded off against additional marine feeding. More research on this neglected topic is needed, given the value of large spring migrating Atlantic salmon to anglers, ongoing declines in their abundance in Ireland and elsewhere, and the widespread occurrence of premature migration in salmonids generally.
The objective of the present study was to determine how low progesterone (P4) affects the endometrial transcriptome, with specific emphasis on those changes that may impact conceptus elongation. ...Following estrous synchronization and detection (estrus = Day 0, n = 40), heifers were randomly assigned to a control group (n = 12) or a low P4 group (n = 28). Heifers in the low P4 group had consistently lower P4 concentrations compared to controls (P < 0.05). Microarray analysis of endometrial gene expression revealed low P4 altered the expression of 498 differentially expressed genes (DEGs; 215 up- and 283 down-regulated) on Day 7 and 351 DEGs (272 up- and 79 down-regulated) on Day 13. A similar number of temporal changes occurred between Day 7 and Day 13 in both groups (2212 in heifers with normal P4 compared with 2247 in heifers with low P4); of these DEGs, 1278 were common to both groups. Little overlap in the number of DEGs affected by high or low P4 was observed across days. Comparison of the temporal changes that occur during normal estrous cycle progression (i.e., from Day 7 to Day 13) to those affected by altered P4 found significant numbers of genes were modulated by elevated (4157) and decreased (809) P4 alone. Analysis of selected genes by quantitative real-time PCR and in situ hybridization revealed that expression of MEP1B, NID2, and PRSS23 increased on Day 13 compared to Day 7 (P < 0.05) and that the magnitude of increase was significantly diminished in heifers with low P4 compared to controls. MEP1B predominantly localized to the both the superficial and deep glandular epithelium (GE), NID2 localized to the deep GE, whereas PRSS23 localized only to the luminal epithelium. In conclusion, we have determined the global changes in the endometrial transcriptome induced by decreasing the output of P4 from the corpus luteum in vivo using a unique animal model. Placing these data into context with previous data in which P4 was supplemented or elevated after ovulation, we have identified a panel of genes that are truly regulated in the endometrium by circulating concentrations of P4 in vivo and that likely impact conceptus elongation.
Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation ...(X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy-a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression.
The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a ...dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I′ domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2·E3BP was thought to be 60 E2 plus ∼12 E3BP. We have prepared homogenous human components. E2 and E2·E3BP have s20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2·E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2·E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ∼60 E1 and maximally sedimented 64.4 ± 1.5 S faster than E2, whereas E1-saturated E2·E3BP maximally sedimented 49.5 ± 1.4 S faster than E2·E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (∼12) were bound by E2·E3BP than by E2. The findings of a smaller E2·E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I′ domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248·E3BP12 mass, which is consistent with this model.
Pyruvate dehydrogenase kinase (PDHK) regulates the activity of the pyruvate dehydrogenase multienzyme complex. PDHK inhibition provides a route for therapeutic intervention in diabetes and ...cardiovascular disorders. We report crystal structures of human PDHK isozyme 2 complexed with physiological and synthetic ligands. Several of the PDHK2 structures disclosed have C-terminal cross arms that span a large trough region between the N-terminal regulatory (R) domains of the PDHK2 dimers. The structures containing bound ATP and ADP demonstrate variation in the conformation of the active site lid, residues 316−321, which enclose the nucleotide β and γ phosphates at the active site in the C-terminal catalytic domain. We have identified three novel ligand binding sites located in the R domain of PDHK2. Dichloroacetate (DCA) binds at the pyruvate binding site in the center of the R domain, which together with ADP, induces significant changes at the active site. Nov3r and AZ12 inhibitors bind at the lipoamide binding site that is located at one end of the R domain. Pfz3 (an allosteric inhibitor) binds in an extended site at the other end of the R domain. We conclude that the N-terminal domain of PDHK has a key regulatory function and propose that the different inhibitor classes act by discrete mechanisms. The structures we describe provide insights that can be used for structure-based design of PDHK inhibitors.
Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy ...(cryo-EM) of the human full-length and truncated E2 (tE2) cores revealed flexible linkers emanating from the edges of trimers of the internal catalytic domains. Using the secondary structure constraints revealed in our 8 Å cryo-EM reconstruction and the prokaryotic tE2 atomic structure as a template, we derived a pseudo atomic model of human tE2. The active sites are conserved between prokaryotic tE2 and human tE2. However, marked structural differences are apparent in the hairpin domain and in the N-terminal helix connected to the flexible linker. These permutations away from the catalytic center likely impart structures needed to integrate a second component into the inner core and provide a sturdy base for the linker that holds the pyruvate dehydrogenase for access by the E2-bound regulatory kinase/phosphatase components in humans.
Purpose: To examine the feasibility for identifying and enumerating cytokeratin positive (CK+) cells in the peripheral blood of breast
cancer patients.
Experimental Design: Blood specimens from 34 ...normal donors (negative controls), 15 samples to which carcinoma cells were added (positive controls),
and 84 breast cancer patients 27 node-negative (N−), 29 node-positive (N+), and 28 metastatic were studied. RBCs were lysed
with ammonium chloride and the resulting cell suspension incubated with anti-EpCAM-conjugated immunomagnetic beads for carcinoma
cell enrichment. Immunomagnetically selected cells were placed on slides; stained for CKs 8, 18, and 19; and evaluated with
an automated digital microscopy system that rapidly scanned the slide and collected images of cells meeting predefined staining
and cytomorphological criteria. A montage of the CK+ cells was reviewed to confirm tumor cell morphology.
Results: Eighteen specimens (9 normal, 2 N−, 4 N+, and 3 metastatic) were excluded because of poor cytomorphology or staining artifact.
All 15 of the positive controls 95% confidence interval (CI), 78–100% and none of the 25 negative controls (95% CI, 0–14%)
demonstrated CK+ cells. Twenty-one of the 75 (28%; 95% CI, 18–40%) samples from breast cancer patients demonstrated CK+ cells
including 76% of patients with metastatic disease (95% CI, 55–91%), 8% with N+ disease (95% CI, 1–26%), and none of those
with N− disease (95% CI, 0–14). The mean number of CK+ cells detected in the 21 CK+ patients was 18.4 (range, 1–120).
Conclusions: Breast carcinoma cells can be detected in the blood from a significant fraction of metastatic breast cancer patients using
immunomagnetic cell enrichment and digital microscopy. The incidence of CK+ cells was low in those with resected N+ disease
(at most 26%) and those with resected N− breast cancer (at most 14%). This technique could be used in large prospective studies
of patients with breast cancer to learn whether the detection of rare carcinoma cells is a useful predictive or prognostic
factor.
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