Double hexameric ring assembly of the type III protein translocase ATPase HrcN

The specialized type III secretion (T3S) apparatus of pathogenic and symbiotic Gram-negative bacteria comprises a complex transmembrane organelle and an ATPase homologous to the F1-ATPase beta subunit. The T3S ATPase HrcN of Pseudomonas syringae associates with the inner membrane, and its ATP hydrolytic activity is stimulated by dodecamerization. The structure of dodecameric HrcN (HrcN12) determined to 1.6 nm by cryo-electron microscopy is presented. HrcN12 comprises two hexameric rings that are probably stacked face-to-face by the association of their C-terminal domains. It is 11.5 +/- 1.0 nm in diameter, 12.0 +/- 2.0 nm high and has a 2.0-3.8 nm wide inner channel. This structure is compared to a homology model based on the structure of the F1-beta-ATPase. A model for its incorporation within the T3S apparatus is presented.     

The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine

The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well-characterized C-terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S-adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV-visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site-directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.     

Bioavailability, antioxidant and immune-enhancing properties of zinc methionine

Although a large number of transition metals and cations remarkably induce oxidative deterioration of biological macromolecules including lipids, proteins and DNA, the trace element zinc acts as a novel dietary supplement and an essential micronutrient, and serves a wide range of biological functions in human and animal health. Zinc promotes antioxidant and immune functions, stabilizes and maintains the structural integrity of biological membranes, and plays a pivotal role in skin and connective tissue metabolism and repair. Zinc is an integral constituent of a large number of enzymes including antioxidant enzymes, and hormones including glucagon, insulin, growth hormone, and sex hormones. High concentrations of zinc are found in the prostate gland and choroids of the eye. Zinc deficiency leads to biochemical abnormalities including the impairments of growth, dermal, gastrointestinal, neurologic and immunologic systems. Given its superior bioavailability, antioxidant and immune-enhancing properties, zinc methionine may serve as a novel dietary supplement to promote health benefits in humans and animals.     

Interpolation techniques to reduce error in measurement of toe clearance during obstacle avoidance

Foot and toe clearance (TC) are used regularly to describe locomotor control for both clinical and basic research. However, accuracy of TC during obstacle crossing can be compromised by typical sample frequencies, which do not capture the frame when the foot is over the obstacle due to high limb velocities. The purpose of this study was to decrease the error of TC measures by increasing the spatial resolution of the toe trajectory with interpolation. Five young subjects stepped over an obstacle in the middle of an 8 m walkway. Position data were captured at 600 Hz as a gold standard signal (GS-600-Hz). The GS-600-Hz signal was downsampled to 60 Hz (DS-60-Hz). The DS-60-Hz was then interpolated by either upsampling or an algorithm. Error was calculated as the absolute difference in TC between GS-600-Hz and each of the remaining signals, for both the leading limb and the trailing limb. All interpolation methods reduced the TC error to a similar extent. Interpolation reduced the median error of trail TC from 5.4 to 1.1 mm; the maximum error was reduced from 23.4 to 4.2 mm (16.6-3.8%). The median lead TC error improved from 1.6 to 0.5 mm, and the maximum error improved from 9.1 to 1.8 mm (5.3-0.9%). Therefore, interpolating a 60 Hz signal is a valid technique to decrease the error of TC during obstacle crossing.     

ZnT3 mRNA levels are reduced in Alzheimer's disease post-mortem brain

Extensive genetic, biochemical, and histological evidence has implicated the amyloid-β peptide (Aβ) in Alzheimer's disease pathogenesis, and several mechanisms have been suggested, such as metal binding, reactive oxygen species production, and membrane pore formation. However, recent evidence argues for an additional role for signaling mediated by the amyloid precursor protein, APP, in part via the caspase cleavage of APP at aspartate 664. Here we review the effects and implications of this cleavage event, and propose a model of Alzheimer's disease that focuses on the critical nature of this cleavage and its downstream effects.     

Effects of extracellular nucleotides on renal tubular solute transport

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y₁ receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K⁺ channels in mouse collecting duct, apparently through stimulation of P2Y₂ receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y₂ receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.     

Functional Interaction between Chfr and Kif22 Controls Genomic Stability

Proper activation of checkpoint during mitotic stress is an important mechanism to prevent genomic instability. Chfr (Check point protein with FHA (Forkhead-associated domain) and RING domains) is a ubiquitin-protein isopeptide ligase (E3) that is important for the control of an early mitotic checkpoint, which delays entry into metaphase in response to mitotic stress. Because several lines of evidence indicate that Chfr is a potential tumor suppressor, it is critically important for us to identify Chfr substrates and understand how Chfr may regulate these substrates, control mitotic transitions, and thus, act as a tumor suppressor in vivo. Here, we report the discovery of a new Chfr-associated protein Kif22, a chromokinesin that binds to both DNA and microtubules. We demonstrated that Kif22 is a novel substrate of Chfr. We showed that Chfr-mediated Kif22 down-regulation is critical for the maintenance of chromosome stability. Collectively, our results reveal a new substrate of Chfr that plays a role in the maintenance of genome integrity.Chfr (Check point protein with FHA and RING domains) is an early mitotic checkpoint protein that delays entry into metaphase in response to mitotic stress (1, 2). The checkpoint function of Chfr requires both of its FHA³ and RING domains. The exact role of FHA domain in Chfr function is largely unknown. Chfr via its RING domain transfers both lysine 48-linked and lysine 63-linked polyubiquitin chains to its target proteins, which either promotes the degradation of target proteins or alters their function (3, 4). Recently, a PAR-binding zinc finger motif, which binds directly to polyADP-ribosylated substrates catalyzed by PARP1, was identified at the C-terminal region of Chfr (5). This PAR-binding zinc finger motif was reported to be required for Chfr function in antephase checkpoint (2, 5).Chfr delays the cell cycle progression at mitosis by inactivating cyclin B1-bound Cdc2 and then exporting them from nucleus (6). Further, mechanistic studies have suggested that the inactivation of Cdc2 may be due to a negative regulation of Plk1 by Chfr (3). Polyubiquitination of Plk1 by Chfr negatively regulates the Plk1 protein levels, which delay the inactivation of Cdc2 inhibitory Wee1 kinase and the activation of Cdc25 phosphatase and thus maintain Cdc2 at its inactive state.Several lines of evidence indicate that Chfr is a potential tumor suppressor. Loss or down-regulation of Chfr has been reported in several types of cancers including primary breast, lung, esophagus, colon, and gastric carcinomas (1, 7, 8). To investigate directly whether Chfr loss contributes to tumorigenesis, our laboratory has generated Chfr knock-out mice, which were cancer-prone and developed spontaneous tumors (9). The increased tumor incidence in Chfr null mice is likely due to a failure in maintaining chromosomal stability, which occurs at least partially due to the overexpression of a key mitotic kinase Aurora A (9). Chfr physically interacts with Aurora A and promotes its ubiquitination and degradation; thus, higher protein levels of Aurora A in Chfr null mice may contribute to chromosomal instability and eventually tumorigenesis. Therefore, our current hypothesis is that Chfr may regulate the stability of several of its substrates including Aurora A, and thus, control mitotic progression and prevent chromosomal instability. In this study, we reported the identification of another Chfr substrate as chromokinesin protein Kif22 and revealed that Kif22 overexpression also contributes to chromosomal instability observed in Chfr-deficient cells.     

A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.     

Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Background  

Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.