Martin Middleditch

Is the failing heart out of fuel or a worn engine running rich? A study of mitochondria in old spontaneously hypertensive rats.

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause‐and‐effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20‐month‐old SHR and Wistar‐Kyoto controls by iTRAQ™‐labelling combined with multidimensional LC/MS/MS. Of 137 high‐scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.

The Structure of MbtI from Mycobacterium tuberculosis, the First Enzyme in the Biosynthesis of the Siderophore Mycobactin, Reveals It To Be a Salicylate Synthase

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 A, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.

Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.

Characterization of proteomic changes in cardiac mitochondria in streptozotocin-diabetic rats using iTRAQ™ isobaric tags.

Diabetes now affects more than 5% of the worlds population and heart failure is the most common cause of death amongst diabetic patients. Accumulating evidence supports a view that myocardial mitochondrial structural and functional changes are central to the onset of diabetic heart failure, but the exact nature of these changes at the proteomic level remains unclear.Here we report on proteomic changes in diabetic rat heart mitochondria following 120 days of streptozotocin‐diabetes using the recently developed iTRAQ™ labeling method, which permits quantification of proteins directly from complex mixtures, bypassing the limitations associated with gel‐based methods such as 2‐DE. Of 252 unique proteins identified, 144 were represented in at least three of six individual paired experiments. Relative amounts of 65 proteins differed significantly between the groups, confirming that the cardiac mitochondrial proteome is indeed impacted by diabetes. The most significant changes were increased protein levels of enzymes involved in mitochondrial oxidation of long‐chain fatty acids, which was also confirmed by enzyme assays, and decreased levels of multiple enzymes involved in oxidative phosphorylation and catabolism of short‐chain fatty acids and branched‐chain amino acids. We also found significant changes in levels of several enzymes linked to oxidative stress.

Proteins Associated with Immunopurified Granules from a Model Pancreatic Islet β-Cell System: Proteomic Snapshot of an Endocrine Secretory Granule

beta-Cell granules contain proteins involved in fuel regulation, which when altered, contribute to metabolic disorders including diabetes mellitus. We analyzed proteins present in purified granules from the INS-1E beta-cell model. Fifty-one component proteins were identified by LC-MS/MS including hormones, granins, protein processing components, cellular trafficking components, enzymes implicated in cellular metabolism and chaperone proteins. These findings may increase understanding of granule secretion and the processes leading to protein aggregation and beta-cell death in type-2 diabetes.

Lower Fetuin-A, Retinol Binding Protein 4 and Several Metabolites after Gastric Bypass Compared to Sleeve Gastrectomy in Patients with Type 2 Diabetes

Background Bypass of foregut secreted factors promoting insulin resistance is hypothesized to be one of the mechanisms by which resolution of type 2 diabetes (T2D) follows roux-en-y gastric bypass (GBP) surgery. Aim To identify insulin resistance-associated proteins and metabolites which decrease more after GBP than after sleeve gastrectomy (SG) prior to diabetes remission. Methods Fasting plasma from 15 subjects with T2D undergoing GBP or SG was analyzed by proteomic and metabolomic methods 3 days before and 3 days after surgery. Subjects were matched for age, BMI, metformin therapy and glycemic control. Insulin resistance was calculated using homeostasis model assessment (HOMA-IR). For proteomics, samples were depleted of abundant plasma proteins, digested with trypsin and labeled with iTRAQ isobaric tags prior to liquid chromatography-tandem mass spectrometry analysis. Metabolomic analysis was performed using gas chromatography-mass spectrometry. The effect of the respective bariatric surgery on identified proteins and metabolites was evaluated using two-way analysis of variance and appropriate post-hoc tests. Results HOMA-IR improved, albeit not significantly, in both groups after surgery. Proteomic analysis yielded seven proteins which decreased significantly after GBP only, including Fetuin-A and Retinol binding protein 4, both previously linked to insulin resistance. Significant decrease in Fetuin-A and Retinol binding protein 4 after GBP was confirmed using ELISA and immunoassay. Metabolomic analysis identified significant decrease of citrate, proline, histidine and decanoic acid specifically after GBP. Conclusion Greater early decrease was seen for Fetuin-A, Retinol binding protein 4, and several metabolites after GBP compared to SG, preceding significant weight loss. This may contribute to enhanced T2D remission observed following foregut bypass procedures.

The proteome of rodent mesenteric lymph

Mesenteric lymph contributes to normal homeostasis and has an emerging role in the pathogenesis of multiple organ dysfunction syndrome. The aim of this study was to define the proteome of normal rodent mesenteric lymph in the fasted and fed states. Eight male Wistar rats fed a standard rodent diet were randomized to two groups. Group 1 (fasted, n = 4) were fasted for 24 h before anesthetized collection of mesenteric lymph. Group 2 (fed, n = 4) were allowed ad libitum access to food before lymph collection. Mesenteric lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry (LC-MS/MS). One hundred fifty proteins, including 26 hypothetical proteins, were identified in this study. All proteins were identified in lymph from both the fasted and fed states. The relative distribution profiles of protein functional classes in the mesenteric lymph differed significantly from that reported for plasma. The most abundant classes identified in lymph were protease inhibitors (16%) and proteins related to innate immunity (12%). In conclusion, this study provides the first detailed description of the normal mesenteric lymph proteome in the fed and fasted states using iTRAQ and LC-MS/MS.

Changes in the mesenteric lymph proteome induced by hemorrhagic shock.

Biologically active factors produced by the intestine and transported by the aqueous and protein fraction of mesenteric lymph are now thought to contribute significantly to the development of distant organ failure in hemorrhagic shock. Despite the likely relevance of the protein composition of mesenteric lymph conditioned by hemorrhagic shock, there is no detailed description of its proteome. The aim of this study was to provide the first comprehensive description of the proteome of hemorrhagic shock-conditioned mesenteric lymph. Mesenteric lymph was collected from 16 male Wistar rats randomized to group 1 (n = 8) sham control and group 2 (n = 8) with hemorrhagic shock. The lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry. Sixty of the 245 proteins had a significant increase in their relative abundance in the hemorrhagic shock group. A bioinformatics approach highlighted the importance of the key gene ontology pathways relating to response to injury and metabolic responses as changing most significantly in shock. Using an interactome, we identified several highly connected proteins: 14-3-3 Zeta, 14-3-3 epsilon, actin, aldolase A, calmodulin, cofilin 1, cystatin C, fatty acid-binding protein 4, profilin 1, prolyl 4-hydrolase, peptidylprolyl isomerase, and transgelin. This study provides the first detailed description of protein changes in hemorrhagic shock-conditioned mesenteric lymph, and using a bioinformatics approach, we identified several targets for possible further research.ABBREVIATIONS-HS-hemorrhagic shock; ML-mesenteric lymph; HS-ML-hemorrhagic shock-conditioned mesenteric lymph; MODS-multiple organ dysfunction syndrome; LC-liquid chromatography; MS-mass spectrometry

Structural Model for Covalent Adhesion of the Streptococcus pyogenes Pilus through a Thioester Bond

Background: Cpa, the pilus adhesin from Streptococcus pyogenes, has an active thioester domain. Results: Cpa has a second thioester domain reactive toward biological amines. Conclusion: The crystal structure of spermidine-bound Cpa provides a model for covalent adhesion. Significance: Thioester domains and covalent adhesion may be common in Gram-positive pathogens. The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we have characterized a second active thioester-containing domain on Cpa, the N-terminal domain of Cpa (CpaN). Expression of CpaN in Escherichia coli gave covalently linked dimers. These were shown by x-ray crystallography and mass spectrometry to comprise two CpaN molecules cross-linked by the polyamine spermidine following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion by covalent bonding to target molecules on host cells that mimics that used by the human complement system to eliminate pathogens.

Quantitative proteomic profiling identifies new renal targets of copper(II)-selective chelation in the reversal of diabetic nephropathy in rats.

This study aimed to identify new diabetic nephropathy (DN)‐related proteins and renal targets of the copper(II)‐selective chelator, triethylenetetramine (TETA) in streptozotocin‐diabetic rats. We used the recently developed iTRAQ™ technology to compare renal protein profiles among non‐diabetic, diabetic, and TETA‐treated diabetic rats. In diabetic kidneys, tubulointerstitial nephritis antigen (TINag), voltage‐dependent anion‐selective channel (VDAC) 1, and VDAC2 were up‐regulated in parallel with alterations in expression of proteins with functions in oxidative stress and oxidative phosphorylation (OxPhos) pathways. By contrast, mitochondrial HSP 60, Cu/Zn‐superoxide dismutase, glutathione S‐transferase α3 and aquaporin‐1 were down‐regulated in diabetic kidneys. Following TETA treatment, levels of D‐amino acid oxidase‐1, epoxide hydrolase‐1, aquaporin‐1, and a number of mitochondrial proteins were normalized, with concomitant amelioration of albuminuria. Changes in levels of TINag, collagen VIα1, actinin 4α, apoptosis‐inducing factor 1, cytochrome C, histone H3, VDAC1, and aquaporin‐1 were confirmed by Western blotting or immunohistochemistry. Changes in expression of proteins related to tubulointerstitial function, podocyte structure, and mitochondrial apoptosis are implicated in the mechanism of DN and their reversal by TETA. These findings are consistent with the hypothesis that this new experimental therapy may be useful for treatment of DN.