Mutual antagonism of target of rapamycin and calcineurin signaling

Growth and stress are generally incompatible states. Stressed cells adapt to an insult by restraining growth, and conversely, growing cells keep stress responses at bay. This is evident in many physiological settings, including for example, the effect of stress on the immune or nervous system, but the underlying signaling mechanisms mediating such mutual antagonism are poorly understood. In eukaryotes, a central activator of cell growth is the protein kinase target of rapamycin (TOR) and its namesake signaling network. Calcineurin is a conserved, Ca(2+)/calmodulin-dependent protein phosphatase and target of the immunosuppressant FK506 (tacrolimus) that is activated in yeast during stress to promote cell survival. Here we show yeast mutants defective for TOR complex 2 (TORC2) or the essential homologous TORC2 effectors, SLM1 and SLM2, exhibited constitutive activation of calcineurin-dependent transcription and actin depolarization. Conversely, cells defective in calcineurin exhibited SLM1 hyperphosphorylation and enhanced interaction between TORC2 and SLM1. Furthermore, a mutant SLM1 protein (SLM1(DeltaC14)) lacking a sequence related to the consensus calcineurin docking site (PxIxIT) was insensitive to calcineurin, and SLM1(Delta)(C14) slm2 mutant cells were hypersensitive to oxidative stress. Thus, TORC2-SLM signaling negatively regulates calcineurin, and calcineurin negatively regulates TORC2-SLM. These findings provide a molecular basis for the mutual antagonism of growth and stress

La dinámica de los estados de fosforilación de las vías HOG y de las feromonas en levaduras

Comunicaciones a congreso

Insights from yeast into whether the rapamycin inhibition of heat shock transcription factor (Hsf1) can prevent the Hsf1 activation that results from treatment with an Hsp90 inhibitor

In human cells TORC1 mTOR (target of rapamycin) protein kinase complex renders heat shock transcription factor 1 (Hsf1) competent for stress activation. In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 “bypass” strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9 – the latter the ortholog of human S6 protein kinase 1. Significance: A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a “pro-survival” role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved

Molecular pathogenesis and targeted therapy of sporadic pancreatic neuroendocrine tumors

Over the past few years, knowledge regarding the molecular pathology of sporadic pancreatic neuroendocrine tumors (PNETs) has increased substantially, and a number of targeted agents have been tested in clinical trials in this tumor type. For some of these agents there is a strong biological rationale. Among them, the mammalian target of rapamycin inhibitor Everolimus and the antiangiogenic agent Sunitinib have both been approved for the treatment of PNETs. However, there is lack of knowledge regarding biomarkers able to predict their efficacy, and mechanisms of resistance. Other angiogenesis inhibitors, such as Pazopanib, inhibitors of Src, Hedgehog or of PI3K might all be useful in association or sequence with approved agents. On the other hand, the clinical significance, and potential for treatment of the most common mutations occurring in sporadic PNETs, in the MEN-1 gene and in ATRX and DAXX, remains uncertain. The present paper reviews the main molecular changes occurring in PNETs and how they might be linked with treatment options

The Synergistic Effect of SAHA and Parthenolide in MDA-MB231 Breast Cancer Cells

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015

Chemical-Biology-derived in vivo Sensors: Past, Present, and Future.

To understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000's of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to 'omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology

Ptc6 is required for proper rapamycin-induced down-regulation of the genes coding for ribosomal and rRNA processing proteins in S. cerevisiae

Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li+. Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic geneti

Assembloid Agency: Unreal Engine API for brain-on-a-chip platforms

Assembloid Agency proposes an open-source Unreal Engine API for interfacing with brains-on-chips (Amirifar et al. 2022), where the API mediates between living neurons cultured on high-density microelectrode arrays and simulated game environments. Building on “Organoid Array Computing: The Design Space of Organoid Intelligence” (Leung, Loewith, and Frisch 2025), which speculates on a future where three-dimensional brain cultures assemble into more complex cognitive infrastructures and hence may become increasingly ‘designable’ and ‘playable’ organisms (Bongard and Levin 2021). We extend this design space to apply games-design principles to context engineering for organoid intelligence, treating organoids as polycomputational agents trained through reinforcement learning (Smirnova et al. 2023). Our proposed plugin exposes functions for stimulation, recording, visualization, and real-time control of neuronal cultures within Unreal Engine. This allows researchers to prototype experimental contexts rapidly, whether using biological wetware, spiking neural networks, or EEG stand-ins. Game templates will be built on Unreal’s Learning Agents plugin to support single- and multi-organoid training scenarios. Theoretically, the API acts as a mediating membrane where biological and algorithmic agencies “contaminate” each other (Parisi 2013), dissolving model/reality divides and enabling distributed cognition across neurons, code, and simulated space (Hutchins 1996). We address risks such as value capture by incorporating reward-schema versioning, rotating reward schedules, and stress monitoring (Nguyen 2024). Assembloid Agency embraces the dual embodiment of biological intelligence. Here, “assembloid” refers to assembling organoids into new computational ecologies, while “agency” invokes not only the game engine as an experimental agent, but also the negotiated play of agency between biological and synthetic actors. By designing an API for game engines, we also anticipate the possibilities of interdisciplinary applications across games, interactive experiences, AI benchmarking, to architectural design, while foregrounding ethical and aesthetic guardrails organoid intelligence