Insight into blocking heme transfer by exploiting molecular interactions in the core Isd heme transporters IsdA-NEAT, IsdC-NEAT, and IsdE of Staphylococcus aureus

The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The nine cell wall and membrane-associated iron regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG and IsdI) allow Staphylococcus aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, it is IsdE that chaperones the heme to the ATP binding cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA and IsdC contain at least one heme binding Near Transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr - proximal amino acid) → IsdC-NEAT (Tyr) → IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. In this paper we investigated PPIX transfer through the core cell wall proteins of the Isd system (IsdA-NEAT, IsdC-NEAT and IsdE) with FePPIX-dimethylester, and the metal substituted CoPPIX and MnPPIX using ESI-MS, UV-visible absorption and MCD spectroscopy. IsdA binds each of the rings but the subsequent transfer properties to IsdC-N or IsdE are not the same as found with heme. FePPIX-DME transfers from IsdA-N to IsdC-N but neither protein transfers the ring to IsdE. IsdA-N does not transfer CoPPIX to IsdC-N or IsdE. IsdA-N does transfer MnPPIX to both IsdC-N and IsdE. Significantly, it is possible that since CoPPIX and FePPIX-DME bind to IsdA-N, the lack of transfer to IsdC-N and subsequently to IsdE for CoPPIX could prove to be used as a potential disruption agent to the S. aureus heme transfer system and may identify a possible anti-microbial.     

Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS would greatly facilitate studies of neuronal network and biomarker discovery.     

Modeling ion channel regulation

Remarkable recent successes in structure determinations of voltage-gated channels, ligand-gated channels, mechanosensitive channels and proton channels have advanced our understanding of the molecular basis of ion channel gating substantially. Models have helped to clarify aspects of this process and are now being designed as sophisticated biomimetics for various technological applications.     

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.     

In vitro characterization of AtsB, a radical SAM formylglycine-generating enzyme that contains three [4Fe-4S] clusters

Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5'-deoxyadenosyl 5'-radical that is generated by a reductive cleavage of S-adenosyl- l-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in iron-sulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 +/- 0.4 irons and 12.2 +/- 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] (2+) configuration, as determined by Mossbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 +/- 0.2 irons and 9.9 +/- 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mossbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys --> Ala substitutions at each of the cysteines in its CX 3CX 2C radical SAM motif contained 7.3 +/- 0.1 irons and 7.2 +/- 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 +/- 0.1 irons and 8.4 +/- 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mossbauer spectra of both samples indicated the presence of only [4Fe-4S] clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max/[E T] of approximately 0.36 min (-1) for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of approximately 0.039 min (-1) for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5'-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is changed to cysteine also gives rise to turnover, supporting approximately 4-fold the activity of that observed with the natural substrate.     

Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis

Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.     

Conformational analysis of the carboxy-terminal tails of human beta-tubulin isotypes

Several isotypes of the structural protein tubulin have been characterized. Their expression offers a plausible explanation for differences regarding microtubule function. Although sequence variation between tubulin isotypes occurs throughout the entire protein, it is the extreme carboxy-terminal tails (CTTs) that exhibit the greatest concentration of differences. In humans, the CTTs range in length from 9 to 25 residues and because of a considerable number of glutamic acid residues, contain over 1/3 of tubulin's total electrostatic charge. The CTTs are believed to be highly disordered and their precise function has yet to be determined. However, their absence has been shown to result in altered microtubule stability and a reduction in the interaction with several microtubule-associated proteins (MAPs). To characterize the role that CTTs play in microtubule function, we examined the global conformational differences within a set of nine human β-tubulin isotypes using replica exchange molecular dynamics simulations. Through the analysis of the resulting configuration ensembles, we quantified differences such as the CTTs sequence influence on overall flexibility and average secondary structure. Although only minor variations between each CTT were observed, we suggest that these differences may be significant enough to affect interactions with MAPs, thereby influencing important properties such as microtubule assembly and stability.     

Reduced ileal expression of OSTalpha-OSTbeta in non-obese gallstone disease

Cholelithiasis is a multifactorial process, and several mechanisms have been postulated. A decreased expression of the ileal apical sodium-dependent bile acid transporter (ASBT) and of the cytosolic ileal lipid binding protein (ILBP) was recently described in female non-obese patients. The role of the recently identified organic solute transporters alpha and beta (OSTalpha, OSTbeta) in gallstone pathogenesis remains unclear. Therefore, we performed analysis of OSTalpha-OSTbeta in gallstone patients according to body weight. Ileal mucosal biopsies were collected during routine colonoscopy from female gallstone carriers (n = 19) and controls (n = 34). OSTalpha-OSTbeta mRNA expression was measured using the LightCycler sequence detection system; protein was analyzed by immunohistochemistry and Western blot. The mRNA expression of OSTalpha-OSTbeta was significantly reduced (OSTalpha: 3.3-fold, P = 0.006; OSTbeta: 2.6-fold, P = 0.03) in normal-weight but not overweight gallstone carriers compared with controls. OSTalpha-OSTbeta protein levels also showed a reduction by 40-67%. The expression of OSTalpha-OSTbeta correlated positively with ASBT (r = 0.65, 0.58, respectively), ILBP (r = 0.77, 0.67), and the farnesoid X receptor (r = 0.58, 0.50). Fibroblast growth factor-19 showed a 2.8-fold reduction (P = 0.06), and liver receptor homolog-1 showed a 2-fold reduction (P = 0.04) in non-obese patients. In conclusion, an impaired function of all three ileal bile acid transporters may lead to low ileal bile acid reabsorption and an altered bile acid pool composition and therefore may contribute to the formation of gallstones in non-obese patients.