Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.     

Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

Molecular, morphological, and ecological niche differentiation of sympatric sister oak species, Quercus virginiana and Q. geminata (Fagaceae)

The genus Quercus (the oaks) is notorious for interspecific hybrization, generating questions about the mechanisms that permit coexistence of closely related species. Two sister oak species, Quercus virginiana and Q. geminata, occur in sympatry in Florida and throughout the southeastern United States. In 11 sites from northern and southeastern regions of Florida, we used a leaf-based morphological index to identify individuals to species. Eleven nuclear microsatellite markers significantly differentiated between the species with a high correspondence between molecular and morphological typing of specimens. Nevertheless, Bayesian clustering analysis indicates interspecific gene flow, and six of 109 individuals had mixed ancestry. The identity of several individuals also was mismatched using molecular markers and morphological characters. In a common environment, the two species performed differently in terms of photosynthetic performance and growth, corresponding to their divergent ecological niches with respect to soil moisture and other edaphic properties. Our data support earlier hypotheses that divergence in flowering time causes assortative mating, allowing these ecologically distinct sister species to occur in sympatry. Limited gene flow that permits ecological differentiation helps to explain the overdispersion of oak species in local communities.     

Phylogenetic community structure in Minnesota oak savanna is influenced by spatial extent and environmental variation

The relative importance of environmental filtering, biotic interactions and neutral processes in community assembly remains an openly debated question and one that is increasingly addressed using phylogenetic approaches. Closely related species may occur together more frequently than expected (phylogenetic clustering) if environmental filtering operates on traits with significant phylogenetic signal. Recent studies show that phylogenetic clustering tends to increase with spatial scale, presumably because greater environmental variation is encompassed at larger spatial scales, providing opportunities for species to sort across environmental gradients. However, if environmental filtering is the cause of species sorting along environmental gradients, then environmental variation rather than spatial scale per se should drive the processes governing community assembly. Using species abundance and light availability data from a long‐term experiment in Minnesota oak savanna understory communities, we explicitly test the hypothesis that greater environmental variation results in greater phylogenetic clustering when spatial scale is held constant. Concordant with previous studies, we found that phylogenetic community structure varied with spatial extent. At the landscape scale (~1000 ha), communities were phylogenetically clustered. At the local scale (0.375ha), phylogenetic community structure varied among plots. As hypothesized, plots encompassing the greatest environmental variation in light availability exhibited the strongest phylogenetic clustering. We also found strong correlations between species functional traits, particularly specific leaf area (SLA) and perimeter per area (PA), and species light availability niche. There was also a phylogenetic signal in both functional traits and species light availability niche, providing a mechanistic explanation for phylogenetic clustering in relation to light availability. We conclude that the pattern of increased phylogenetic clustering with increased environmental variation is a consequence of environmental filtering acting on phylogenetically conserved functional traits. These results indicate that the importance of environmental filtering in community assembly depends not on spatial scale per se, but on the steepness of the environmental gradient.     

Seasonal Variability of Diazotroph Assemblages Associated with the Rhizosphere of the Salt Marsh Cordgrass, Spartina alterniflora

Nitrogen fixation is the primary N source in the highly productive but N-limited North Inlet, SC, USA salt marsh system. The diverse assemblages of nitrogen-fixing (diazotrophic) bacteria associated with the rhizospheres of the short and tall growth forms of Spartina alterniflora were analyzed at two sites, Crab Haul Creek and Goat Island, which are in different tidal creek drainage systems in this marsh. The sites differed in proximity to the main channel for tidal intrusion and in several edaphic parameters. We hypothesized that either the differing abiotic environmental regimes of the two sites or the variation due to seasonal effects result in differences in the diazotroph assemblage. Rhizosphere samples were collected seasonally during 1999 and 2000. DNA was purified and nifH amplified for denaturing gradient gel electrophoresis (DGGE) analysis of diazotroph assemblage composition. Principal components analysis was used to analyze the binary DGGE band position data. Season strongly influenced assemblage composition and biplots were used to identify bands that significantly affected the seasonal and site-specific clustering. The types of organisms that were most responsive to seasonal or site variability were identified on the basis of DGGE band sequences. Seasonally responsive members of the anaerobic diazotrophs were detected during the winter and postsenescence conditions and may have been responsible for elevated pore water sulfide concentrations. Sequences from a diverse assemblage of Gammaproteobacteria were predominant during growth periods of S. alterniflora. Abiotic environmental parameters strongly influenced both the S. alterniflora and the diazotrophic bacterial assemblages associated with this keystone salt marsh plant species.     

Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8^G116 shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.     

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged (“homeologous”) sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally ≤100 bp, even in Msh2^−/− cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.The ability of a cell to repair DNA damage is integral to maintaining genome integrity. One common type of damage that is particularly detrimental is a double-strand break (DSB), where both strands of DNA are broken. If not accurately repaired, DSBs can lead to cell death, chromosomal rearrangements, and loss of genetic material (reviewed in references 14 and 19). One mechanism of DSB repair is homologous recombination (HR), in which an unbroken homologous sequence, the donor of genetic information, is used as a template for repair of the broken sequence, the recipient of genetic information. HR intermediates possess heteroduplex DNA (hDNA), where one strand of DNA is derived from the donor sequence, and the second strand is derived from the recipient sequence. Mismatches in hDNA are substrates of the mismatch repair machinery (MMR) (reviewed in reference 38), leading to gene conversion. HR is the preferred repair pathway of DSBs in Saccharomyces cerevisiae (reviewed in references 42 and 46), plays an important role in repair of DSBs in Drosophila (1, 32), and is a major repair pathway of DSBs that occur during S/G₂ in mammalian cells (33, 54).Two pathways appear to predominate for the repair of DSBs by HR, both of which can give rise to noncrossover products, which predominate in mitotic mammalian cells (Fig. ​(Fig.1)1) (29, 52, 60). In the DSB repair model proposed by Szostak et al. (61), double Holliday junctions are resolved to result in recombinant products (Fig. ​(Fig.1A).1A). More recent evidence suggests the existence of an alternative pathway, termed synthesis-dependent strand annealing (SDSA) (Fig. ​(Fig.1B)1B) (20, 40, 42, 52). One difference between these two pathways is that the DSB repair model requires capture of both DNA ends (Fig. ​(Fig.1A),1A), which can lead to bidirectional gene conversion tracts. In contrast, SDSA can involve only one end of the broken DNA followed by dissociation (Fig. ​(Fig.1B),1B), resulting in predominantly unidirectional gene conversion tracts. Another difference is that the donor sequence can be altered during DSB repair while it typically remains unchanged after SDSA.Open in a separate windowFIG. 1.Models for noncrossover gene conversion resulting from DSB repair. DSB repair is initiated by resection of the DNA ends (black; strand directionality is designated a 3′ “tail”). The resected 3′ overhang invades the homologous donor template (gray), forming hDNA at the site of invasion (i), which acts as a primer/template for repair synthesis (gray dotted line). (A) In the canonical DSB repair (DSBR) model, the second strand of the DSB is captured, resulting in another stretch of hDNA (ii) and repair synthesis, to form a double Holliday junction. Depending on how the double Holliday junction is cleaved (arrowheads), resolution can result in a crossover (data not shown) or a noncrossover, as shown. (B) In SDSA, the newly synthesized strand dissociates from the D-loop and anneals to the other DNA end to form another stretch of hDNA (iii). Repair synthesis and ligation result in a noncrossover product. While one-end invasion is illustrated for the SDSA model, it is possible for both DNA ends to invade, resulting in gene conversion on both sides of the DSB (data not shown). In both models, hDNA formed by the newly synthesized strands can be repaired by MMR, resulting in gene conversion of markers (data not shown).HR repair is sensitive to differences between the recombining sequences, and cells have developed ways to suppress recombination between diverged sequences. This suppression of “homeologous” recombination reduces HR both between diverged repeats and with foreign DNA. Suppression of homeologous recombination is conserved across species and requires the MMR machinery (7, 10, 11, 49, 56). For example, MSH2 dramatically reduces both gene targeting (12) and DSB-induced HR (15) between sequences with >1% divergence in murine embryonic stem (ES) cells.Another protein that has been proposed to suppress homeologous recombination is Sgs1, the budding yeast RecQ helicase, as sequence divergence has little effect on recombination frequencies in Sgs1 mutants (39, 59). Sgs1 mutants have other phenotypes as well; for example, they demonstrate a hyperrecombination phenotype associated with spontaneous repair (22, 65, 68). The mammalian homolog of Sgs1 is BLM, mutants of which also have a hyperrecombination phenotype, as evidenced by a high frequency of sister-chromatid exchange (SCE) in both human and mouse cells (18, 24, 34, 69). Evidence suggests that Drosophila BLM, like Sgs1, has a role in the suppression of homeologous recombination (30) although mammalian BLM has not been tested in this regard. Supporting a possible role for BLM in suppressing homeologous recombination is the observation that BLM associates with MMR factors in a large protein complex (64; reviewed in reference 21), and BLM directly interacts with two components of the MMR machinery, MLH1 (45) and MSH6 (44), which, like MSH2, is known to suppress homeologous recombination (13).To gain more insight into mammalian HR mechanisms, as well as factors that control recombination between homeologous sequences, we examined recombination between homologous and homeologous sequences in both murine and human cells. By taking advantage of multiple, single base pair polymorphisms distributed along the donor in gene conversion substrates, we examined both the nature of gene conversion tracts and the fate of the donor sequence. Unidirectional tracts with a bias in conversion to one side of the DSB predominated in both mouse and human cells, supporting an SDSA mechanism of HR. Moreover, the donor remained unaltered after HR. Interestingly, while transformed human cells suppressed homeologous recombination, the degree of suppression was less than that observed in mouse cells. For either cell type, BLM deficiency did not alter this suppression, unlike what is observed in yeast Sgs1 mutants. Either other RecQ helicase family members play a role in the suppression of homeologous recombination, or mammalian RecQ helicases do not play a role in this process.     

ErbB3/HER3 does not homodimerize upon neuregulin binding at the cell surface

To understand signaling by the neuregulin (NRG) receptor ErbB3/HER3, it is important to know whether ErbB3 forms homodimers upon ligand binding. Previous biophysical studies suggest that the ErbB3 extracellular region remains monomeric when bound to NRG. We used a chimeric receptor approach to address this question in living cells, fusing the extracellular region of ErbB3 to the kinase-active intracellular domain of ErbB1. The ErbB3/ErbB1 chimera responded to NRG only if ErbB2 was co-expressed in the same cells, whereas an ErbB4/ErbB1 chimera responded without ErbB2. We, therefore, suggest that ErbB3 is an obligate heterodimerization partner because of its inability to homodimerize.     

ACE and non-ACE mediated effect of angiotensin I on intracellular calcium mobilization in rat glomerular arterioles

Because renin and angiotensin I (ANG I) level are high in the renal circulation, the conversion of ANG I is a critical step in the regulation of glomerular hemodynamics. We studied this conversion by investigating the effect of ANG I on intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat juxtamedullary glomerular afferent and efferent arterioles (AA and EA, respectively). Two types of EA were considered, thin EA and muscular EA, terminating as peritubular capillaries and vasa rectae, respectively. In all arterioles, ANG I elicited [Ca(2+)](i) elevations. Maximal responses of 171 +/- 28 (AA), 183 +/- 7 (muscular EA), and 78 +/- 11 nM (thin EA) (n = 6), similar to those obtained with ANG II, were observed with 100 nM ANG I. The EC(50) values were 20 times higher for ANG I than for ANG II in AA (10.2 vs. 0.5) and muscular EA (6.8 vs. 0.4 nM) and 150 times higher in thin EA (15.2 vs. 0.1 nM). ANG I effect was blocked by losartan, indicating that AT(1) receptors were involved. The ANG-converting enyzme (ACE) inhibitor lisinopril inhibited the maximal response to ANG I in AA and muscular EA by 75 +/- 9% (n = 13) and 70 +/- 7% (n = 13), respectively, but had no effect in thin EA (n = 14). The serine protease inhibitor aprotinin, the chymase inhibitor chymostatin, and the cysteine protease inhibitors E64 and leupeptin had no effect on ANG I action. These data show that ANG I effects are mainly mediated by ACE in AA and muscular EA but not in thin EA. The lisinopril-insensitive response may be related to conversion by unknown enzyme(s) and/or to activation of AT(1) receptors by ANG I.