Structural insights into SAM domain‐mediated tankyrase oligomerization

Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP‐ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain‐mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head‐to‐tail polymer that facilitates TNKS self‐association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM‐TNKS2 SAM) hetero‐oligomeric structures mediated by their SAM domains. Though wild‐type tankyrase proteins have very low solubility, model‐based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP‐ribosyl)ation (PARylation) and PARylation‐dependent ubiquitylation.     

In vitro poly(ADP‐ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP‐ribosyl)ated in vitro. The modifying product, poly(ADP‐ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis‐specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP‐ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP‐ribose)polymerase in the presence of [³²P] NAD. In parallel, poly(ADP‐ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [³²P] NAD. In both experiments, the poly(ADP‐ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP‐ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10–12 units long, whereas longer chains (≤20 ADP‐R units) were linked to H1t. Individual poly(ADP‐ribosyl)ated H1s were complexed with homologous H1‐depleted oligonucleosomes (0.5–2.5 kbp) in order to measure their ability to condensate chromatin, in comparison with the native ones. Circular dichroism showed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA‐binding properties of the analyzed H1s. In particular, the poly(ADP‐ribosyl)ated H1a and H1t had opposite effects on the condensation of H1‐depleted oligonucleosomes. J. Cell. Biochem. 76:20–29, 1999. © 1999 Wiley‐Liss, Inc.     

Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

Plum pox virus capsid protein suppresses plant pathogen‐associated molecular pattern (PAMP)‐triggered immunity

The perception of pathogen‐associated molecular patterns (PAMPs) by immune receptors launches defence mechanisms referred to as PAMP‐triggered immunity (PTI). Successful pathogens must suppress PTI pathways via the action of effectors to efficiently colonize their hosts. So far, plant PTI has been reported to be active against most classes of pathogens, except viruses, although this defence layer has been hypothesized recently as an active part of antiviral immunity which needs to be suppressed by viruses for infection success. Here, we report that Arabidopsis PTI genes are regulated upon infection by viruses and contribute to plant resistance to Plum pox virus (PPV). Our experiments further show that PPV suppresses two early PTI responses, the oxidative burst and marker gene expression, during Arabidopsis infection. In planta expression of PPV capsid protein (CP) was found to strongly impair these responses in Nicotiana benthamiana and Arabidopsis, revealing its PTI suppressor activity. In summary, we provide the first clear evidence that plant viruses acquired the ability to suppress PTI mechanisms via the action of effectors, highlighting a novel strategy employed by viruses to escape plant defences.     

Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity

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Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.     

A plant effector‐triggered immunity signaling sector is inhibited by pattern‐triggered immunity

Since signaling machineries for two modes of plant‐induced immunity, pattern‐triggered immunity (PTI) and effector‐triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta. These observations led us to discovery of a network‐buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI‐Mediating and PTI‐Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.     

Clustering of multi‐domain protein sequences

The overall function of a multi‐domain protein is determined by the functional and structural interplay of its constituent domains. Traditional sequence alignment‐based methods commonly utilize domain‐level information and provide classification only at the level of domains. Such methods are not capable of taking into account the contributions of other domains in the proteins, and domain‐linker regions and classify multi‐domain proteins. An alignment‐free protein sequence comparison tool, CLAP (CLAssification of Proteins) was previously developed in our laboratory to especially handle multi‐domain protein sequences without a requirement of defining domain boundaries and sequential order of domains. Through this method we aim to achieve a biologically meaningful classification scheme for multi‐domain protein sequences. In this article, CLAP‐based classification has been explored on 5 datasets of multi‐domain proteins and we present detailed analysis for proteins containing (1) Tyrosine phosphatase and (2) SH3 domain. At the domain‐level CLAP‐based classification scheme resulted in a clustering similar to that obtained from an alignment‐based method. CLAP‐based clusters obtained for full‐length datasets were shown to comprise of proteins with similar functions and domain architectures. Our study demonstrates that multi‐domain proteins could be classified effectively by considering full‐length sequences without a requirement of identification of domains in the sequence.     

Protective effects of polyphenolics in red wine on diabetes associated oxidative/nitrative stress in streptozotocin‐diabetic rats

Increased accumulation of NT (3‐nitrotyrosine) and PARylated [poly(ADP‐ribosyl)ated] proteins in the tissues of diabetics are associated with diabetes complications (diabetes neuropathy, nephropathy and retinopathy). Red wine (its polyphenols are considered to be the main active components) can act as ROS (reactive oxygen species) scavengers, iron chelators and enzyme modulators. This study is novel in investigating the effect of red wine in preventing the accumulation of NT and PARylated proteins in the sciatic nerve, DRG (dorsal root ganglia), spinal cord, kidney and retina of diabetic animals. We have shown that during the experiment the body weight of control and diabetic groups of rats with consumption of red wine was significantly increased, by 52% and 19% accordingly. The significant increase in the content of NT in the sciatic nerve, DRG, spinal cord, kidney and retina, and PARylated proteins in the sciatic nerve, renal glomeruli and retinae of diabetic rats was partly or completely prevented by treatment with red wine. Red wine and its polyphenol preparations might be a promising option in the prevention and treatment of diabetic complications.     

High levels of cyclic‐di‐GMP in plant‐associated Pseudomonas correlate with evasion of plant immunity

The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour.     

Spatiotemporal regulation of posttranslational modifications in the DNA damage response

A timely and accurate cellular response to DNA damage requires tight regulation of the action of DNA damage response (DDR) proteins at lesions. A multitude of posttranslational modifications (PTMs) of chromatin and chromatin‐associated proteins coordinates the recruitment of critical proteins that dictate the appropriate DNA repair pathway and enable the actual repair of lesions. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP‐ribosyl)ation, acetylation, and methylation are among the DNA damage‐induced PTMs that have taken center stage as important DDR regulators. Redundant and multivalent interactions of DDR proteins with PTMs may not only be a means to facilitate efficient relocalization, but also a feature that allows high temporal and spatial resolution of protein recruitment to, and extraction from, DNA damage sites. In this review, we will focus on the complex interplay between such PTMs, and discuss the importance of their interconnectivity in coding DNA lesions and maintaining the integrity of the genome.     

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.     

The chromatin-remodeling protein BAF60/SWP73A regulates the plant immune receptor NLRs

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Poly (ADP‐ribose) polymerase‐1 binds to BCL2 major breakpoint region and regulates BCL2 expression

BCL2, originally identified as a proto‐oncogene in B‐cell lymphoma, is a key regulator of apoptosis. Although it is more than 200 kb in length, at least 70% of the t(14;18) translocation in follicular lymphomas occurs at the BCL2 major breakpoint region (mbr), located in the 3′‐untranslated region (3'‐UTR). We have previously found that the mbr is a regulatory element which positively regulates BCL2 expression and this regulatory function was closely associated with SATB1, which binds to a 37 bp mbr (37 mbr) in the 3′‐end of the mbr directly. However, the precise molecular mechanisms by which the mbr regulates gene expression are not fully understood. In this study, we purified Poly(ADP‐ribose) polymerase‐1 (PARP‐1) from the DNA–protein complexes formed by 37 mbr in Jurkat cells and demonstrated that PARP‐1 participates in the 37 mbr–protein complex's formation in vitro and in vivo. Functional analysis showed that overexpression of PARP‐1 decreases 37 mbr regulatory function and BCL2 expression. Conversely, knockdown of PARP‐1 with RNAi increases BCL2 expression. Taken together, the present findings indicate that PARP‐1 is a component of BCL2 37 mbr–protein complexes, and PARP‐1 is involved in the regulation of BCL2 expression. These findings are helpful in understanding the regulatory mechanisms of BCL2 expression. J. Cell. Biochem. 110: 1208–1218, 2010. Published 2010 Wiley‐Liss, Inc.     

Cotton LIM domain‐containing protein GhPLIM1 is specifically expressed in anthers and participates in modulating F‐actin

As one form of actin binding protein (ABP), LIM domain protein can trigger the formation of actin bundles during plant growth and development. In this study, a cDNA (designated GhPLIM1) encoding a LIM domain protein with 216 amino acid residues was identified from a cotton flower cDNA library. Quantitative RT‐PCR indicated that GhPLIM1 is specifically expressed in cotton anthers, and its expression levels are regulated during anther development of cotton. GhPLIM1:eGFP transformed cotton cells display a distributed network of eGFP fluorescence, suggesting that GhPLIM1 protein is mainly localised to the cell cytoskeleton. In vitro high‐speed co‐sedimentation and low co‐sedimentation assays indicate that GhPLIM1 protein not only directly binds actin filaments but also bundles F‐actin. Further biochemical experiments verified that GhPLIM1 protein can protect F‐actin against depolymerisation by Lat B. Thus, our data demonstrate that GhPLIM1 functions as an actin binding protein (ABP) in modulating actin filaments in vitro, suggesting that GhPLIM1 may be involved in regulating the actin cytoskeleton required for pollen development in cotton.     

Regulation of cytoskeleton‐associated protein activities: Linking cellular signals to plant cytoskeletal function

The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associatedproteins,microtubuleassociated proteins(MAPs) and actin-binding proteins(ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.