Characterization of a legumain/vacuolar processing enzyme and YVADase activity in Papaver pollen

Legumains, also known as Vacuolar Processing Enzymes (VPEs) have received considerable attention recently, as they share structural properties with mammalian caspase-1 and exhibit YVADase/caspase-1-like cleavage activity. Although many legumains have been cloned, knowledge about their detailed characteristics and intracellular localization is relatively limited. We previously identified several caspase-like activities activated by self-incompatibility (SI) in pollen; a DEVDase was required for programmed cell death (PCD), but YVADase was not (Bosch and Franklin-Tong in Proc Natl Acad Sci USA 104:18327–18332, 2007; Thomas and Franklin-Tong in Nature 429:305–309, 2004). Here we report identification of a legumain/VPE from Papaver rhoeas pollen (PrVPE1) that binds to the DEVD tetrapeptide, a signature substrate for caspase-3. A detailed characterization of the recombinant PrVPE1 cleavage activity revealed that, like other VPEs, it has YVADase activity and requires an acidic pH for activity. Unlike other legumain/VPEs, it also exhibits DEVDase and IETDase activities and apparently does not require processing for activity. The pollen-expressed PrVPE1 localizes to a reticulate compartment resembling the vacuole. Examination of YVADase activity using live-cell imaging of pollen tubes revealed YVADase activity in mitochondria of growing pollen tubes. The unexpected features of PrVPE1, together with evidence for YVADase activity in plant mitochondria, indicate that VPEs, YVADases, their localization and functions in plant cells merit further investigation.     

Crystal structure of the C‐terminal domain of the Salmonella type III secretion system export apparatus protein InvA

InvA is a prominent inner‐membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N‐terminal integral membrane domain and a C‐terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C‐terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V‐type ATPase and a ring building motif found in other T3SS proteins respectively.     

BIODIVERSITY RESEARCH: Turning up the heat: the impacts of Andropogon gayanus (gamba grass) invasion on fire behaviour in northern Australian savannas

Aim This study aimed to quantify changes in fire severity resulting from the invasion of Australia’s tropical savannas by the African grass Andropogon gayanus Kunth. (gamba grass). Location Mesic savannas of the Northern Territory, Australia. Method Byram’s fire‐line intensity (I_f), fuel load and architecture, and two post‐fire indicators of fire intensity – scorch height (SH) and char height (CH) of woody vegetation – were determined for fires in native grass savanna and A. gayanus invaded savanna. Leaf scorch is the height at which the fire’s radiant heat browns leaf tissue, and leaf char is the height that radiant heat blackens or consumes leaf tissue and provides an indirect measure of flame height. These data, and 5 years of similar data collected from the Kapalga Fire Project in Kakadu National Park, were used to develop empirical relationships between I_f and the post‐fire indices of fire intensity. Results A relationship between A. gayanus I_f and SH could not be developed because complete canopy scorch occurred in most A. gayanus fires, even at low I_f. In contrast, A. gayanus I_f was strongly correlated with CH. This empirical relationship was substantially different from that for native grass fires. For a given I_f, there was a significantly greater CH in invaded sites. This increase in radiant heat is attributable to the increased biomass (mean 3.6 t ha^?1 in native grasses compared to 11.6 t ha^?1 in A. gayanus) and height (approximately 0.5 m in native grasses compared to 4 m in A. gayanus) of the standing fine fuel. Main conclusion Andropogon gayanus invasion resulted in substantial changes in fire behaviour. This has important regional implications owing to the current (10,000–15,000 km²) and predicted (380,000 km²) area of invasion and the negative consequences for the native savanna biota that has evolved with frequent but relatively low‐intensity fire.     

Cell‐Free Reconstitution of Multivesicular Body Formation and Receptor Sorting

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell‐free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal‐transducing adaptor molecule (STAM), or addition of mutated ATPase‐deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.     

Different regulation of cigarette smoke induced inflammation in upper versus lower airways

Background

Cigarette smoke (CS) is known to initiate a cascade of mediator release and accumulation of immune and inflammatory cells in the lower airways. We investigated and compared the effects of CS on upper and lower airways, in a mouse model of subacute and chronic CS exposure.

Methods

C57BL/6 mice were whole-body exposed to mainstream CS or air, for 2, 4 and 24 weeks. Bronchoalveolar lavage fluid (BAL) was obtained and tissue cryosections from nasal turbinates were stained for neutrophils and T cells. Furthermore, we evaluated GCP-2, KC, MCP-1, MIP-3α, RORc, IL-17, FoxP3, and TGF-β1 in nasal turbinates and lungs by RT-PCR.

Results

In both upper and lower airways, subacute CS-exposure induced the expression of GCP-2, MCP-1, MIP-3α and resulted in a neutrophilic influx. However, after chronic CS-exposure, there was a significant downregulation of inflammation in the upper airways, while on the contrary, lower airway inflammation remained present. Whereas nasal FoxP3 mRNA levels already increased after 2 weeks, lung FoxP3 mRNA increased only after 4 weeks, suggesting that mechanisms to suppress inflammation occur earlier and are more efficient in nose than in lungs.

Conclusions

Altogether, these data demonstrate that CS induced inflammation may be differently regulated in the upper versus lower airways in mice. Furthermore, these data may help to identify new therapeutic targets in this disease model.     

Identification of evolutionarily conserved genetic regulators of cellular aging

To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.     

Phosphorylation of p22phox on Threonine 147 Enhances NADPH Oxidase Activity by Promoting p47phox Binding

NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events. Previously, our laboratory demonstrated that the small membrane subunit of the oxidase complex, p22^phox, is phosphorylated in neutrophils and that its phosphorylation correlates with NADPH oxidase activity. In this study, we utilized site-directed mutagenesis in a Chinese hamster ovarian cell system to determine the phosphorylation sites within p22^phox. We also explored the mechanism by which p22^phox phosphorylation affects NADPH oxidase activity. We found that mutation of threonine 147 to alanine inhibited superoxide production in vivo by more than 70%. This mutation also blocked phosphorylation of p22^phox in vitro by both protein kinase C-α and -δ. Moreover, this mutation blocked the p22^phox-p47^phox interaction in intact cells. When phosphorylation was mimicked in vivo through mutation of Thr-147 to an aspartyl residue, NADPH oxidase activity was recovered, and the p22^phox-p47^phox interaction in the membrane was restored. Maturation of gp91^phox was not affected by the alanine mutation, and phosphorylation of the cytosolic component p47^phox still occurred. This study directly implicates threonine 147 of p22^phox as a critical residue for efficient NADPH oxidase complex formation and resultant enzyme activity.     

Bax Contains Two Functional Mitochondrial Targeting Sequences and Translocates to Mitochondria in a Conformational Change- and Homo-oligomerization-driven Process

The apoptosis gateway protein Bax normally exists in the cytosol as a globular shaped monomer composed of nine α-helices. During apoptosis, Bax translocates to the mitochondria, forms homo-oligomers, and subsequently induces mitochondrial damage. The mechanism of Bax mitochondrial translocation remains unclear. Among the nine α-helices of Bax, helices 4, 5, 6, and 9 are capable of targeting a heterologous protein to mitochondria. However, only helices 6 and 9 can independently direct the oligomerized Bax to the mitochondria. Although Bax mitochondrial translocation can still proceed with mutations in either helix 6 or helix 9, combined mutations completely abolished mitochondrial targeting in response to activating signals. Using a proline mutagenesis scanning analysis, we demonstrated that conformational changes were sufficient to cause Bax to move from the cytosol to the mitochondria. Moreover, we found that homo-oligomerization of Bax contributed to its mitochondrial translocation. These results suggest that Bax is targeted to the mitochondria through the exposure of one or both of the two functional mitochondrial targeting sequences in a conformational change-driven and homo-oligomerization-aided process.     

Solution luminescence from chloro(2,2′:6′,2″-terpyridine)platinum(II) chloride in micelles

Previously characterized as being non-luminescent in room-temperature fluid solution, the coordination compound chloro(2,2′:6′,2″-terpyridine)platinum(II) chloride can display two types of luminescence in certain microenvironments. In aqueous solutions of anionic and neutral surfactants having concentrations near or above their critical micelle concentration, [Pt(terpy)Cl]Cl (10-50 μM) displays broad emission centered at ∼610 nm that is characterized as metal-to-ligand charge transfer phosphorescence (³MLCT). In high concentration (10-100 mM) solutions having no surfactant, [Pt(terpy)Cl]Cl aggregates form. Excitation in the 470-540 nm region results in a long-wavelength emission centered at ∼720 nm that is characterized as metal-metal-to-ligand charge transfer phosphorescence (³MMLCT). This emission can also be detected in lower concentration solutions (10-50 μM) with surfactant concentration below its critical micelle concentration. Enhancement of ³MLCT luminescence is also found for the related phenylacetylide complex cation [Pt(terpy)(CCPh)]⁺ in micelles of the anionic surfactant sodium dodecyl sulfate.     

Genome sequence of the obligate methanotroph Methylosinus trichosporium strain OB3b

Methylosinus trichosporium OB3b (for "oddball" strain 3b) is an obligate aerobic methane-oxidizing alphaproteobacterium that was originally isolated in 1970 by Roger Whittenbury and colleagues. This strain has since been used extensively to elucidate the structure and function of several key enzymes of methane oxidation, including both particulate and soluble methane monooxygenase (sMMO) and the extracellular copper chelator methanobactin. In particular, the catalytic properties of soluble methane monooxygenase from M. trichosporium OB3b have been well characterized in context with biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of the M. trichosporium OB3b genome is the first reported from a member of the Methylocystaceae family in the order Rhizobiales.