SUMMARYArabidopsis 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°C and exposure to 45°C. Transgenic ROF1 over-expressors showed better survival in response to exposure to 45°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°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.
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.
Background. There has been a significant increase in the use of immunotherapy and cannabis recently, two modalities that have immunomodulatory effects and may have possible interaction. We evaluated the influence of cannabis use during immunotherapy treatment on response rate (RR), progressionfree survival (PFS), and overall survival (OS). Subjects, Materials, and Methods. In this retrospective, observational study, data were collected from the files of patients treated with nivolumab in the years 2015-2016 at our hospital, and cannabis from six cannabis-supplying companies. Included were 140 patients (89 nivolumab alone, 51 nivolumab plus cannabis) with advanced melanoma, non-small cell lung cancer, and renal clear cell carcinoma. The groups were homogenous regarding demographic and disease characteristics. A comparison between the two arms was made.Results. In a multivariate model, cannabis was the only significant factor that reduced RR to immunotherapy (37.5% RR in nivolumab alone compared with 15.9% in the nivolumab-cannabis group (p = .016, odds ratio = 3.13, 95% confidence interval 1.24-8.1). Cannabis use was not a significant factor for PFS or OS. Factors affecting PFS and OS were smoking (adjusted hazard ratio [HR] = 2.41 and 2.41, respectively (and brain metastases (adjusted HR = 2.04 and 2.83, respectively). Low performance status (adjusted HR = 2.83) affected OS alone. Tetrahydrocannabinol and cannabidiol percentages did not affect RR in any group (p = .393 and .116, respectively). Conclusion. In this retrospective analysis, the use of cannabis during immunotherapy treatment decreased RR, without affecting PFS or OS and without relation to cannabis composition. Considering the limitations of the study, further prospective clinical study is needed to investigate possible interaction. The Oncologist 2019;24:549-554 Implications for Practice: Although the data are retrospective and a relation to cannabis composition was not detected, this information can be critical for cannabis users and indicates that caution is required when starting immunotherapy.
Cannabis or its derivatives are widely used by patients with cancer to help with cancer symptoms and treatment side effects. However, cannabis has potent immunomodulatory properties. To determine if cannabis consumption during immunotherapy affects therapy outcomes, we conducted a prospective observatory study including 102 (68 immunotherapy and 34 immunotherapy plus cannabis) consecutive patients with advanced cancers who initiated immunotherapy. Cannabis consumption correlated with a significant decrease in time to tumor progression and overall survival. On the other hand, the use of cannabis reduced therapy-related immune-related adverse events. We also tested the possibility that cannabis may affect the immune system or the tumor microenvironment through the alteration of the endocannabinoid system. We analyzed a panel of serum endocannabinoids (eCBs) and eCB-like lipids, measuring their levels before and after immunotherapy in both groups. Levels of serum eCBs and eCB-like lipids, before immunotherapy, showed no significant differences between cannabis users to nonusers. Nevertheless, the levels of four eCB and eCB-like compounds were associated with patients’ overall survival time. Collectively, cannabis consumption has considerable immunomodulatory effects, and its use among cancer patients needs to be carefully considered due to its potential effects on the immune system, especially during treatment with immunotherapy.
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.
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.
The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant’s therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-Δ 9 - trans -tetrahydrocannabinol (Δ 9 -THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of Δ 9 -THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract’s composition as well as on certain characteristics of the targeted cells.
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 phosphorylationdependent 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.RhoGTPases are key regulators of transcription, cell cycle progression, and the organization of the microtubule and actin cytoskeletons. By cycling between active GTP-bound and inactive GDP-coupled states, these enzymes behave as molecular switches. The activation state of RhoGTPases is governed by the balance between the activities of GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs). While the hydrolysis of GTP to GDP by RhoGTPases is enhanced by RhoGAPs, RhoGEFs mediate the exchange of GDP for GTP.Characterized by tandem Dbl homology (DH) and pleckstrin homology (PH) domains, the Dbl family represents the largest group of RhoGEFs. The DH domain mediates binding to inactive GTPases and catalyzes the exchange of GDP for GTP. The role of the PH domain is less well defined and may facilitate the interaction of some RhoGEFs with the plasma membrane and cooperate with the DH domain in activating RhoGTPases (45). In addition to the DH-PH core, many RhoGEFs also possess extended N and/or C termini with negative regulatory functions. Thus, a number of RhoGEFs are constitutively activated by N-or C-terminal truncation (21,34,36). Moreover, N and C termini frequently mediate interactions with other proteins, thereby functioning to integrate several signaling pathways. The regulator of G protein signaling (RGS) homology domain-containing RhoGEFs, p115RhoGEF (35), LARG (50), and PDZ-RhoGEF (25), for instance, can bind directly to and be activated by the G␣ subunits of heterotrimeric G proteins. Nearly 40% of human Dbl family RhoGEFs contain C-terminal PDZ binding motifs, suggesting that interactions with PDZ domain-containing proteins represent a common mechanism for controlling RhoGEF localization and activity (26). A number of RhoGEFs possess unrelated domains in addition to the tandem DH-PH core, thus allowing the enzymes to nucleate unique signaling networks. For instance, mammalian Son-of-sevenless (Sos) can coordinate the activities of both Rac and Ras by virtue of both a tandem DH-PH cassette and a RasGEF homology domain (12, 42). Kalirin and Trio have separate functional GEF domains for Rho and Rac in ad...
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