David Meiri

Arabidopsis ROF1 (FKBP62) modulates thermotolerance by interacting with HSP90.1 and affecting the accumulation of HsfA2-regulated sHSPs.

Arabidopsis ROF1 (AtFKBP62) is a peptidyl prolyl cis/trans isomerase and a member of the FKBP (FK506 binding protein) family. ROF1 expression is induced by heat stress and developmentally regulated. In this study, we show that ROF1 binds heat shock proteins HSP90.1 via its tetratricopeptide repeat domain, and localizes in the cytoplasm under normal conditions. Exposure to heat stress induces nuclear localization of the ROF1-HSP90.1 complex, which is dependent upon the presence of the transcription factor HsfA2, which interacts with HSP90.1 but not with ROF1. Nuclear localization of ROF1 was not detected in Arabidopsis HSP90.1 and HsfA2 knockout mutants. The rof1 knockout plants exhibited collapse when 24-48 h passed between acclimation at 37 degrees C and exposure to 45 degrees C. Transgenic ROF1 over-expressors showed better survival in response to exposure to 45 degrees C than wild-type plants did. In rof1 knockout mutants, the level of expression of small HSPs regulated by HsfA2 was dramatically reduced after exposure to 37 degrees C and recovery for 24-48 h, and correlates well with the mutant phenotype. We suggest a role for ROF1 in prolongation of thermotolerance by sustaining the levels of small HSPs that are essential for survival at high temperatures.

Involvement of Arabidopsis ROF2 (FKBP65) in thermotolerance

The ROF2 (FKBP65) is a heat stress protein which belongs to the FK506 Binding Protein (FKBP) family. It is homologous to ROF1 (FKBP62) which was recently shown to be involved in long term acquired thermotolerance by its interaction with HSP90.1 and modulation of the heat shock transcription factor HsfA2. In this study, we have demonstrated that ROF2 participates in long term acquired thermolerance, its mode of action being different from ROF1. In the absence of ROF2, the small heat shock proteins were highly expressed and the plants were resistant to heat stress, opposite to the effect observed in the absence of ROF1. It was further demonstrated that ROF2 transcription is modulated by HsfA2 which is also essential for keeping high levels of ROF2 during recovery from heat stress. ROF2 localization to the nucleus was observed several hours after heat stress exposure and its translocation to the nucleus was independent from the presence of HSP90.1 or HsfA2. ROF2 has been shown to interact with ROF1, to form heterodimers and it is suggested that via this interaction it can join the complex ROF1-HSP90.1- HsfA2. Transient expression of ROF2 together with ROF1 repressed transcription of small HSPs. A model describing the mode of action of ROF2 as a heat stress modulator which functions in negative feedback regulation of HsfA2 is proposed.

Sumoylation of Arabidopsis heat shock factor A2 (HsfA2) modifies its activity during acquired thermotholerance.

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulate many cellular processes. In this work we show SUMOylation of the heat shock transcription factor, AtHsfA2, in connection with the plant’s response to heat stress and acquired thermotolerance. Using the Yeast two hybrid and the bimolecular fluorescence complementation system, we have found that AtSUMO1 physically interacts with AtHsfA2. Further investigation allowed us to determine that Lys 315 of AtHsfA2 is the main SUMOylation site. Overexpression of AtSUMO1 led to a decrease in AtHsfA2 transcriptional activation of heat shock promoters. We have examined the effect of AtSUMO1 on AtHsfA2 during heat shock treatments. The phenotype of seedlings overexpressing AtSUMO1 resembled the phenotype of AtHsfA2 knock out seedlings, which were more sensitive than wild type seedlings to repeated heat treatment. Furthermore, AtSUMO1 overexpressing seedlings exhibited lower expression levels of small heat shock proteins as compared with wild type seedlings after heat treatment. Based on our findings, we suggest that AtSUMO1 is involved in the regulation of AtHsfA2 in acquired thermotolerance.

Mechanistic Insight into the Microtubule and Actin Cytoskeleton Coupling through Dynein-Dependent RhoGEF Inhibition

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.

Modulation of Rho Guanine Exchange Factor Lfc Activity by Protein Kinase A-Mediated Phosphorylation

ABSTRACT Lfc is a guanine nucleotide exchange factor (GEF) for Rho that demonstrates an unusual ability to associate with microtubules. While several phosphorylated residues have been detected in the Lfc polypeptide, the mechanism(s) by which phosphorylation regulates the exchange activity of Lfc remains unclear. We confirm that Lfc is a phosphorylated protein and demonstrate that 14-3-3 interacts directly and in a phosphorylation-dependent manner with Lfc. We identify AKAP121 as an Lfc-binding protein and show that Lfc is phosphorylated in an AKAP-dependent manner by protein kinase A (PKA). Forskolin treatment induced 14-3-3 binding to Lfc and suppressed the exchange activity of wild-type Lfc on RhoA. Importantly, a mutant of Lfc that is unable to associate with 14-3-3 proteins was resistant to inhibition by forskolin. Tctex-1, a dynein motor light chain, binds to Lfc in a competitive manner with 14-3-3.

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gβγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gβγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation.

RhoGTPases – A novel link between cytoskeleton organization and cisplatin resistance

For more than three decades, platinum compounds have been the first line treatment for a wide spectrum of solid tumors. Yet, cisplatin resistance is a major impediment in cancer therapy, and deciphering the mechanisms underlying chemoresistance is crucial for the development of novel therapies with enhanced efficacy. The Rho subfamily of small GTPases plays a significant role in cancer progression, and a growing body of evidence points toward the involvement of these proteins in anticancer drug resistance, including cisplatin resistance. The cycling between active and inactive states, governed by the balance between their GEFs, GAPs and GDIs, RhoGTPases, acts as molecular switches with a pivotal role in actin cytoskeleton organization. The Rho subfamily of proteins is involved in many key cellular processes including adhesion, vesicular trafficking, proliferation, survival, cell morphology and cell-matrix interactions. Although RhoA, RhoB and RhoC are highly homologous and share some upstream regulators and downstream effectors, they each have different roles in cancer progression and chemoresistance. While RhoA and RhoC are upregulated in many tumors and can stimulate transformation, RhoB appears to exhibit tumor suppressor characteristics with proapoptotic effects. In the current review, we discuss the role of Rho subfamily of proteins in cancer, and focus on their involvement in intrinsic and acquired drug resistance.

Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts.

The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.

A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis

Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study’s approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.