Inhibition Of Protein Synthesis Research Articles

Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.

Pathologic α-synuclein plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. We report that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD.

Enhanced Ferroptosis Induction Susceptibility in Diffuse Large B-Cell Lymphoma through Rocaglate-Mediated Translation Inhibition

Introduction: Diffuse Large B-cell Lymphoma (DLBCL) is a genomically complex hematologic malignancy characterized by heterogeneous pathogenesis across multiple molecular subtypes. Despite recent advances, standard frontline treatment options leave a third of patients with relapsed or refractory disease due to tumor populations resistant to therapy by a wide variety of mechanisms. Ferroptosis, an iron-dependent non-programmed cell death mechanism driven by lipid peroxidation, has emerged as a promising therapeutic opportunity in DLBCL. DLBCL demonstrates distinct sensitivity to ferroptosis activators compared to other cancer types, yet the implications in DLBCL prognosis, immune infiltration, and drug resistance remain elusive. Notably, the loss of histone deubiquitinase MYSM1 in hematopoietic stem cells significantly reduces protein synthesis, leading to reduced ferroptosis suppressors, ultimately rendering the cells more susceptible to ferroptosis. Moreover, NRF2, encoded by NFE2L, a known modulator of ferroptosis, has been linked to resistance to rocaglates (cap-dependent translation inhibitors). Intriguingly, our preliminary data indicate that rocaglates induce translation of key proteins involved in oxidative stress (i.e. SDHB) and transcription factors related to the oxidative stress response (NRF1, ATF2, NRF2). Additionally, our published findings in glioblastoma suggest the upregulation of the ferroptosis suppressor 4F2 ( SLC3A2) under translation inhibition conditions. Furthermore, our investigations uncover another potential mechanism, wherein rocaglates induce NF-κB, which was previously shown to be suppressed by dimethyl fumarate (DMF) upon ferroptosis induction in DLBCL. However, the intricate interplay between ferroptosis and translation inhibition in DLBCL remains uninvestigated, prompting our hypothesis that protein synthesis inhibitors might enhance therapeutic ferroptosis induction. Method: To test our hypothesis, we conducted a comprehensive analysis, employing TMT-pSILAC to assess the global transcriptome response to rocaglates treatment, and subsequently verified significant findings via western blot analysis. Additionally, we quantified reduced glutathione (GSH) levels, reactive oxygen species (ROS) levels, and synergy with ferroptosis inducers (e.g., erastin, RSL3) to elucidate the mechanism of rocaglates in enhancing susceptibility to the ferroptosis pathway. Moreover, we explored NF-κB, previously identified as a ferroptosis regulator in our RNAseq analysis, using ELISA and NF-κB reporter assays. Results: Our TMT-pSILAC analysis unveiled upregulation of key ferroptosis suppressors, specifically NFS1, CBS, and 4F2. Concurrent treatment with rocaglates resulted in a notable increase in GSH levels, which was attenuated when administered in combination with ferroptosis inducers (p-value < 0.005) concomitant with the reduction of Glutathione Peroxidase 4 (GPX4), modulator of lipid peroxidation. Although rocaglates alone did not elicit significant levels of reactive oxygen species (ROS), their combination with ferroptosis inducers resulted in dramatic elevation of ROS levels (p-value < 0.0005). Correspondingly, there was strong synergy between the clinical rocaglate zotatifin and different ferroptosis inducers (Bliss δ synergy score > 10) and antagonism with ferroptosis inhibitors (Bliss δ synergy score < -10). Furthermore, our investigation revealed a noteworthy upregulation of NFKB1 at the translatome level, accompanied by a significant increase in TNF-α expression at 48 hours. The NF-κB-EGFP reporter assay confirmed the enhanced expression of NF-κB in response to the treatment. Conclusion: In conclusion, rocaglate-induced stress upregulates multiple ferroptosis suppressors, suggesting that cap-dependent translation disruption triggers a protective response against ferroptosis. Strikingly, these factors did not protect DLBCL tumors from ferroptosis inducers, which synergized remarkably with rocaglates, yielding promising new combination drug strategies. The proteomic basis for ferroptosis sensitization by rocaglate therapy remains under active investigation. Understanding the intricate interplay between these pathways may unveil potential key mechanisms for DLBCL treatment, potentially offering novel therapeutic targets to improve patient outcomes.

Repositioning of mifepristone as an integrated stress response activator to potentiate cisplatin efficacy in non-small cell lung cancer

Lung cancer, primarily non-small-cell lung cancer (NSCLC), is a significant cause of cancer-related mortality worldwide. Cisplatin-based chemotherapy is a standard treatment for NSCLC; however, its effectiveness is often limited due to the development of resistance, leading to NSCLC recurrence. Thus, the identification of effective chemosensitizers for cisplatin is of paramount importance. The integrated stress response (ISR), activated by various cellular stresses and mediated by eIF2α kinases, has been implicated in drug sensitivity. ISR activation globally suppresses protein synthesis while selectively promoting the translation of ATF4 mRNA, which can induce pro-apoptotic proteins such as CHOP, ATF3, and TRIB3. To expedite and economize the development of chemosensitizers for cisplatin treatment in NSCLC, we employed a strategy to screen an FDA-approved drug library for ISR activators. In this study, we identified mifepristone as a potent ISR activator. Mifepristone activated the HRI/eIF2α/ATF4 axis, leading to the induction of pro-apoptotic factors, independent of its known role as a synthetic steroid. Our in vitro and in vivo models demonstrated mifepristone's potential to inhibit NSCLC re-proliferation following cisplatin treatment and tumor growth, respectively, via the ISR-mediated cell death pathway. These findings suggest that mifepristone, as an ISR activator, could enhance the efficacy of cisplatin-based therapy for NSCLC, highlighting the potential of drug repositioning in the search for effective chemosensitizers.

Improved capacity for the repair of photosystem II via reinforcement of the translational and antioxidation systems in Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803, translation factor EF-Tu is inactivated by reactive oxygen species (ROS) via oxidation of Cys82 and the oxidation of EF-Tu enhances the inhibition of the repair of photosystem II (PSII) by suppressing protein synthesis. In our present study, we generated transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together. Expression of EF-Tu (C82S) alone in Synechocystis enhanced the repair of PSII under strong light, with the resultant mitigation of PSII photoinhibition, but it stimulated the production of ROS. However, overexpression of superoxide dismutase and catalase, together with the expression of EF-Tu (C82S), lowered intracellular levels of ROS and enhanced the repair of PSII more significantly under strong light, via facilitation of the synthesis de novo of the D1 protein. By contrast, the activity of photosystem I was hardly affected in wild-type cells and in all the lines of transformed cells under the same strong-light conditions. Furthermore, transformed cells that overexpressed EF-Tu (C82S), superoxide dismutase, and catalase were able to survive longer under stronger light than wild-type cells. Thus, the reinforced capacity for both protein synthesis and ROS scavenging allowed both photosynthesis and cell proliferation to tolerate strong light.

KCNH2A561V Heterozygous Mutation Inhibits KCNH2 Protein Expression via The Activation of UPR Mediated by ATF6.

The potassium channel protein KCNH2 is encoded by KCNH2 gene, and there are more than 300 mutations of KCNH2. Unfolded protein response (UPR) is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER). The present study aimed to explore the UPR process and the role of activating transcription factor 6 (ATF6) in the abnormal expression of potassium voltage-gated channel subfamily H member 2 (KCNH2)A561V. The wild-type (wt) KCNH2 and A561V mutant KCNH2 was constructed with his-tag. The 293 cells were used and divided into KCNH2wt+KCNH2A561V, KCNH2wt and KCNH2A561V groups. The expression levels of ATF6 and KCNH2 in different groups were detected by Western blotting, reverse transcription-quantitative PCR, immunofluorescence and immuno-coprecipitation assays. The protein types and abundance of immuno-coprecipitation samples were analyzed by mass spectrometry. The proteomic analysis of the mass spectrometry results was carried out by using the reactome database and GO (Gene Ontology) tool. The mRNA expression levels of KCNH2 and ATF6 in the KCNH2wt+KCNH2A561V group were higher compared with the KCNH2A561V group. However, the full-length protein expression of ATF6 was inhibited, indicating that ATF6 was highly activated and a substantial number of ATF6 was sheared in KCNH2wt+KCNH2A561V group compared with control group. Furthermore, A561V-KCNH2 mutation leading to the accumulation of the immature form of KCNH2 (135 kDa bands) in ER, resulting in the reduction of the ratio of 155 kDa/135 kDa. In addition, the abundance of UPR-related proteins in the KCNH2A561V group was higher compared with the KCNH2wt+KCNH2A561V group. The 'cysteine biosynthetic activity' of GO:0019344 process and the 'positive regulation of cytoplasmic translation activity' of GO:2000767 process in the KCNH2A561V group were higher compared with the KCNH2wt+KCNH2A561V group. Hence, co-expression of wild-type and A561V mutant KCNH2 in 293 cells activated the UPR process, which led to the inhibition of protein translation and synthesis, in turn inhibiting the expression of KCNH2. These results provided a theoretical basis for clinical treatment of Long QT syndrome.

RNF144B-mediated p21 degradation regulated by HDAC3 contribute to enhancing ovarian cancer growth and metastasis

We have shown before that HDAC3 was involved in the pathogenesis of ovarian cancer; however, the specific mechanism of HDAC3 on the pathogenesis of ovarian cancer has not been thoroughly studied. To explore the related proteins in the mechanism of HDAC3 on ovarian cancer. The transcriptome profiles were identified in ovarian carcinoma cells with HDAC3 knockdown or overexpression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to verify transfection efficiency. Immunofluorescence staining were performed to detect the expression levels of HDAC3 and RNF144B in tissues. Cell proliferation, apoptosis, migration and invasion were confirmed by cell counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase (TUNEL) and transwell assay, respectively. The protein expression of p53, p21, Bax and Bcl-2 was confirmed by western blot, and CoIP assay was used to validate RNF144B/P21/P53 interaction. Meanwhile, the protein synthesis inhibitor cycloheximide (CHX) was performed to treat cells to probe p21 stability. Finally, we established an in vivo tumor model to explore the effects of HDAC3 and RNF144B on tumor growth. Microarray results showed that among the overlapping genes in the two profiles (HDAC3 knockdown and overexpression), RNF144B was decreased or increased in ovarian carcinoma cells with HDAC3 knockdown or overexpression, HDAC3 overexpression promoted RNF144B expression, and HDAC3 knockdown hindered RNF144B levels. The levels of HDAC3 and RNF144B in malignant ovarian cancer were significantly higher than those in normal ovarian tissue and benign ovarian cancer tissue. RNF144B promoted cell proliferation, migration and invasion, and inhibited cell apoptosis. In addition, overexpression of HDAC3 or RNF144B inhibited p53/p21/Bax expression and promoted Bcl-2 expression. Knockout of HDAC3 or RNF144B has the opposite effect, and RNF144B interacted with p21 and regulated the p21/p53 complex degradation, and finally in vivo experiments proved that HDAC3 and RNF144B promoted tumor growth. RNF144B-mediated p21 degradation regulated by HDAC3 contributed to enhancing ovarian cancer growth and metastasis.

A meta-analysis of the effect of protein synthesis inhibitors on rodent fear conditioning

A meta-analysis of the effect of protein synthesis inhibitors on rodent fear conditioning

Birinapant selectively enhances immunotoxin-mediated killing of cancer cells conditional on the IAP protein levels within target cells.

Immunotoxins (ITs) target cancer cells via antibody binding to surface antigens followed by internalization and toxin-mediated inhibition of protein synthesis. The fate of cells responding to IT treatment depends on the amount and stability of specific pro-apoptotic and pro-survival proteins. When treated with a pseudomonas exotoxin-based immunotoxin (HB21PE40), the triple-negative breast cancer (TNBC) cell line MDA-MB-468 displayed a notable resistance to toxin-mediated killing compared to the epidermoid carcinoma cell line, A431, despite succumbing to the same level of protein synthesis inhibition. In a combination screen of ~1912 clinically relevant and mechanistically annotated compounds, we identified several agents that greatly enhanced IT-mediated killing of MDA-MB-468 cells while exhibiting only a modest enhancement for A431 cells. Of interest, two Smac mimetics, birinapant and SM164, exhibited this kind of differential enhancement. To investigate the basis for this, we probed cells for the presence of inhibitor of apoptosis (IAP) proteins and monitored their stability after the addition of immunotoxin. We found that high levels of IAPs inhibited immunotoxin-mediated cell death. Further, TNFα levels were not relevant for the combination's efficacy. In tumor xenograft studies, combinations of immunotoxin and birinapant caused complete regressions in MDA-MB-468tumor-bearing mice but not in mice with A431 tumors. We propose that IAPs constitute a barrier to immunotoxin efficacy which can be overcome with combination treatments that include Smac mimetics.

Theoretical study of the interaction between the antibiotic linezolid and the active site of the 50S ribosomal subunit of the bacterium Haloarcula marismortui.

First antibiotic in the oxazolidinone class, linezolid fights gram-positive multiresistant bacteria by inhibiting protein synthesis through its interaction with the 50S subunit of the functional bacterial ribosome. For its antimicrobial action, it is necessary that its chiral carbon located in the oxazolidinone ring is in the S-conformation. Computational calculation at time-dependent density functional theory methodology, ultraviolet-visible (UV-Vis), and electronic circular dichroism spectra was obtained for noncomplexed and complexed forms of linezolid to verify the possible chirality of nitrogen atom in the acetamide group of the molecule. The molecular system has two chiral centers. So, there are now four possible configurations: RR, RS, SR, and SS. For a better understanding of the system, the electronic spectra at the PBE0/6-311++G(3df,2p) level of theory were obtained. The complexed form was obtained from the crystallographic data of the ribosome, containing the S-linezolid molecular system. The computational results obtained for the electronic properties are in good agreement with the experimental crystallographic data and available theoretical results.

Diaphorin, a polyketide produced by a bacterial endosymbiont of the Asian citrus psyllid, adversely affects the in vitro gene expression with ribosomes from Escherichia coli and Bacillus subtilis.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria), an obligate mutualist of an important agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera). Our previous study demonstrated that diaphorin, at physiological concentrations in D. citri, inhibits the growth and cell division of Bacillus subtilis (Firmicutes) but promotes the growth and metabolic activity of Escherichia coli (Gammaproteobacteria). This unique property of diaphorin may aid microbial mutualism in D. citri, potentially affecting the transmission of "Candidatus Liberibacter spp." (Alphaproteobacteria), the pathogens of the most destructive citrus disease Huanglongbing. Moreover, this property may be exploited to promote microbes' efficiency in producing industrial materials. However, the mechanism underlying this activity is unknown. Diaphorin belongs to the family of pederin-type compounds, which inhibit protein synthesis in eukaryotes by binding to eukaryotic ribosomes. Therefore, as a first step to assess diaphorin's direct influence on bacterial gene expression, this study examined the effect of diaphorin on the in vitro translation using ribosomes of B. subtilis and E. coli, quantifying the production of the green fluorescent protein. The results showed that the gene expression involving B. subtilis and E. coli ribosomes along with five millimolar diaphorin was 29.6% and 13.1%, respectively, less active than the control. This suggests that the diaphorin's adverse effects on B. subtilis are attributed to, at least partly, its inhibitory effects on gene expression. Moreover, as ingredients of the translation system were common other than ribosomes, the greater inhibitory effects observed with the B. subtilis ribosome imply that the ribosome is among the potential targets of diaphorin. On the other hand, the results also imply that diaphorin's positive effects on E. coli are due to targets other than the core machinery of transcription and translation. This study demonstrated for the first time that a pederin congener affects bacterial gene expression.

Homoharringtonine promotes heart allograft acceptance by enhancing regulatory T cells induction in a mouse model.

Homoharringtonine (HHT) is an effective anti-inflammatory, anti-viral, and anti-tumor protein synthesis inhibitor that has been applied clinically. Here, we explored the therapeutic effects of HHT in a mouse heart transplant model. Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver, kidney, and hematology. A mouse heart transplantation model was constructed, and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan-Meier analysis, immunostaining, and bulk RNA sequencing analysis. The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells (Tregs) differentiation. HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo . Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days ( P <0.001) without non-immune toxicity. The allografts had long-term survival after continuous HHT treatment for 28 days. HHT significantly reduced lymphocyte infiltration in the graft, and interferon-γ-secreting CD4 + and CD8 + T cells in the spleen ( P <0.01). HHT significantly increased the number of peripheral Tregs (about 20%, P <0.001) and serum interleukin (IL)-10 levels. HHT downregulated the expression of T cell receptor (TCR) signaling pathway-related genes ( CD4 , H2-Eb1 , TRAT1 , and CD74 ) and upregulated the expression of IL-10 and transforming growth factor (TGF)-β pathway-related genes and Treg signature genes ( CTLA4 , Foxp3 , CD74 , and ICOS ). HHT increased CD4 + Foxp3 + cells and Foxp3 expression ex vivo , and it enhanced the inhibitory function of inducible Tregs. HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels, thereby promoting mouse heart allograft acceptance. These findings may have therapeutic implications for organ transplant recipients, particularly those with viral infections and malignancies, which require a more suitable anti-rejection medication.

What is Learned Determines How Pavlovian Conditioned Fear is Consolidated in the Brain.

Activity in the basolateral amygdala complex (BLA) is needed to encode fears acquired through contact with both innate sources of danger (i.e., things that are painful) and learned sources of danger (e.g., being threatened with a gun). However, within the BLA, the molecular processes required to consolidate the two types of fear are not the same: protein synthesis is needed to consolidate the first type of fear (so-called first-order fear) but not the latter (so-called second-order fear). The present study examined why first- and second-order fears differ in this respect. Specifically, it used a range of conditioning protocols in male and female rats, and assessed the effects of a BLA infusion of the protein synthesis inhibitor, cycloheximide, on first- and second-order conditioned fear. The results revealed that the differential protein synthesis requirements for consolidation of first- and second-order fears reflect differences in what is learned in each case. Protein synthesis in the BLA is needed to consolidate fears that result from encoding of relations between stimuli in the environment (stimulus-stimulus associations, typical for first-order fear) but is not needed to consolidate fears that form when environmental stimuli associate directly with fear responses emitted by the animal (stimulus-response associations, typical for second-order fear). Thus, the substrates of Pavlovian fear conditioning in the BLA depend on the way that the environment impinges upon the animal. This is discussed with respect to theories of amygdala function in Pavlovian fear conditioning, and ways in which stimulus-response associations might be consolidated in the brain.

Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures.

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.

Exercise attenuates polyglutamine-mediated neuromuscular degeneration in a mouse model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a hereditary neuromuscular disorder caused by the expansion of trinucleotide cytosine-adenine-guanine (CAG) repeats, which encodes a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. Recent evidence suggests that, in addition to motor neuron degeneration, defective skeletal muscles are also the primary contributors to the pathogenesis in SBMA. While benefits of physical exercise have been suggested in SBMA, underlying mechanism remains elusive. We investigated the effect of running exercise in a transgenic mouse model of SBMA carrying human AR with 97 expanded CAGs (AR97Q). We assigned AR97Q mice to exercise and sedentary control groups, and mice in the exercise group received 1-h forced running wheel (5m/min) 5days a week for 4weeks during the early stage of the disease. Motor function (grip strength and rotarod performance) and survival of each group were analysed, and histopathological and biological features in skeletal muscles and motor neurons were evaluated. AR97Q mice in the exercise group showed improvement in motor function (~40% and ~50% increase in grip strength and rotarod performance, respectively, P<0.05) and survival (median survival 23.6 vs. 16.7weeks, P<0.05) with amelioration of neuronal and muscular histopathology (~1.4-fold and ~2.8-fold increase in motor neuron and muscle fibre size, respectively, P<0.001) compared to those in the sedentary group. Nuclear accumulation of polyQ-expanded AR in skeletal muscles and motor neurons was suppressed in the mice with exercise compared to the sedentary mice (~50% and ~30% reduction in 1C2-positive cells in skeletal muscles and motor neurons, respectively, P<0.05). We found that the exercise activated 5'-adenosine monophosphate-activated protein kinase (AMPK) signalling and inhibited mammalian target of rapamycin pathway that regulates protein synthesis in skeletal muscles of SBMA mice. Pharmacological activation of AMPK inhibited protein synthesis and reduced polyQ-expanded AR proteins in C2C12 muscle cells. Our findings suggest the therapeutic potential of exercise-induced effect via AMPK activation in SBMA.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer.

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

The role of the encephalomyocarditis virus type 1 proteins L and 2A in the inhibition of the synthesis of cellular proteins and the accumulation of viral proteins during infection

Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A «security» proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery. To study the role of L and 2A «security» proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells. Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; Δ2A encoding a partially deleted 2A; Zfmut&Δ2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography. The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2α, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation. A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.

STAT5-Feedback Controls Distinct Metabolic States for Dynamic Transitions between Cellular Activation and Quiescence in Acute Lymphoblastic Leukemia

Background and significance: B-cell acute lymphoblastic leukemia (B-ALL) is characterized by dynamic shifts between activation and quiescence. These cycles are predicated by fluctuations in oncogenic signaling, which mimic pre-BCR and cytokine receptor signaling, e.g. IL7 receptor signaling and phosphorylation of STAT5. These fluctuations in cytokine receptor and STAT5 signaling are mitigated by powerful feedback regulators, including CISH and SOCS (suppressors of cytokine signaling) family members. While B-ALL cells depend on oncogenic STAT5 signaling, CISH and SOCS2 negative feedback regulators of STAT5 are highly expressed in aggressive B-ALL and high expression levels are strongly linked to poor clinical outcome, positive MRD and increased likelihood of bone marrow relapse in children (COG P9906) and adults (ECOG E2993). To determine whether high levels of CISH and SOCS2 are biomarkers of high oncogenic STAT5 signaling or mechanistically contribute to clonal fitness and adaptability of B-ALL, we performed genetic experiments in mouse models for B-ALL. Here we examined the mechanisms and consequences of active and inactive states of STAT5 by studying constitutively active (CA) and non-phosphorylatable Y694F (YF) mutations of STAT5. Results: Consistent with a central role of STAT5-feedback control in B-ALL survival, inducible ablation of Cishfl/fl resulted in rapid depletion of B-ALL cells. Socs2-/- B-ALL cells rapidly underwent cellular senescence, lacked colony forming and leukemia-initiating capacity in serial transplantation experiments. Likewise direct interference with STAT5-function, by mutations resulting in constitutive activation (STAT5-CA) or an inactive state (STAT5-YF). Consistent with a requirement of adaptive STAT5-feedback control in B-ALL, both Stat5-CA and Stat5-YF resulted in rapid cell death. Induction of Stat5-CA caused accumulation of biomass indicated by their increased cell size, whereas Stat5-YF caused rapid cell shrinkage ( Figure A). To determine whether the mTOR cell growth pathway was responsible for cell death induced by Stat5-CA, we tested whether the genetic deletion of Mtor or pharmacological inhibition of protein synthesis were sufficient to rescue B-ALL cell death. As expected, B-ALL cells retaining intact Mtor rapidly underwent energy crisis and cell death, while genetic deletion of Mtor reversed the toxic effects of Stat5-CA, suggesting that Stat5-CA is associated with excessive protein synthesis and defective ER turnover. Indeed, Stat5-CA increased protein synthesis rate and ER content compared to EV control, and these effects were opposed by Stat5-YF. To compare the metabolic outcomes of Stat5-CA and Stat5-YF, we performed mass spectrometry-based metabolomic analyses ( Figure B). Consistent with the role of mTOR in promoting glycolysis, Stat5-CA increased the content of glycolytic intermediates in B-ALL cells. In contrast, Stat5-YF increased phospholipid intermediates and phosphatidylethanolamine (PtdEtn). Opposed by Myc, Stat5-YF increased the expression levels of Bcl6 and autophagic genes, suggesting that Bcl6-dependent programs promote PtdEtn-LC3B conjugation, thereby autophagosome formation. As expected, microscopic validation with LC3B-GFP reporter revealed that Stat5-YF increased the number of LC3B-GFP puncta, upon pharmacological inhibition of autophagosome-lysosome fusion ( Figure A). Consistent with STAT5-CA promoting Myc- as opposed to STAT5-YF driving Bcl6-dependent programs, a flow cytometry analysis of Myc-eGFP/Bcl6-mCherry dual reporter expression in murine B-ALL cells demonstrated that changes between glycolytic Myc+ and autophagic Bcl6+ states were the direct consequence of Stat5-CA and Stat5-YF induction, respectively. Conclusions: Our results demonstrate feedback regulators of STAT5, including SOCS2 and CISH are not only biomarkers of increased oncogenic STAT5 activity and a more aggressive course of disease linked to poor overall outcomes. Importantly, negative STAT5 feedback control mechanisms are essential to enable nimble adaptations of B-ALL cells in response to fluctuations of oncogenic signaling strength and availability of nutrients during distinct metabolic states of cellular activation (MYC) and quiescence (BCL6).

Metabolic Reprogramming By PRDM16 Drives Cytarabine Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the underlying molecular mechanisms are poorly understood. Here, we show that AML cells with high PRDM16 expression acquire resistance to cytarabine (AraC) through the activation of oxidative phosphorylation (OxPHOS) via regulation of c-MYC. Initially, we established a murine model of AML with high Prdm16 expression. We transduced c-Kit + bone marrow cells from wild-type mice with a retrovirus vector encoding MLL-AF9 (MF9) and the oncogenic short form of Prdm16 (sPrdm16). Using MF9/sPrdm16 cells, we next investigated whether AML cells with high Prdm16 expression exhibited resistance to AraC and/or daunorubicin (DNR), chemotherapeutic agents commonly used for induction chemotherapy. In the cell viability assay, MF9/sPrdm16 cells showed significantly higher IC 50 against AraC, but not against DNR, compared to MF9/control (control) cells, suggesting that high sPrdm16 expression confers relative resistance to AraC on AML cells. Colony-forming assay also confirmed resistant to AraC in MF9/sPrdm16 cells compared to control. Moreover, MF9/sPrdm16 cells were also resistant to AraC in vivo as demonstrated by the shortened survival of mice receiving MF9/sPrdm16 cells. To elucidate the mechanism of AraC resistance, we first examined cell cycle status of MF9/sPrdm16 cells since AraC exerts cytotoxicity in cell cycle dependent manner. To our surprise, MF9/sPrdm16 cells were cycling more rapidly than control cells as demonstrated by decreased G 0 phase and increased S/G 2/M phase. Intriguingly, RNA-sequencing analysis revealed that MF9/sPrdm16 cells displayed a signature of high OxPHOS by gene set enrichment analysis (GSEA). Indeed, oxygen consumption rate, superoxide production from mitochondria, and mitochondrial membrane potential were significantly elevated in MF9/sPrdm16 cells compared to control cells. The inhibition of mitochondrial respiration with metformin, the electron transport chain complex I inhibitor, or tigecycline, the mitochondrial protein synthesis inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells by inducing an energetic shift towards a lower OxPHOS status. These results clearly indicated that sPrdm16 overexpression conferred MF9 cells with AraC resistance byincreasing OxPHOS in mitochondria. Next, we explored the mechanism how sPrdm16 regulates OxPHOS. In addition to elevated OxPHOS signature, RNA-sequencing data also showed strong enrichment of Myc target-signature in MF9/sPrdm16 cells. Western blot analysis showed that sPrdm16 overexpression induced up-regulation of c-Myc and its transcriptional target, Slc1a5, a glutamine transporter. We went on to analyze metabolic changes induced by sPrdm16 overexpression by capillary electrophoresis and mass spectrometry-based metabolome analysis. We found that sPrdm16 induced the accumulation of glutamine, glutamate, α-ketoglutaric acid, succinate, fumarate, and malate in MF9 cells, possibly by activating glutaminolysis via the regulation of c-Myc and Slc1a5 expression, leading to the activation of tricarboxylic acid (TCA) cycle and OxPHOS. We also confirmed the contribution of c-Myc to AraC resistance by overexpressing c-Myc in MF9 cells. As expected, MF9/c-Myc cells mimicked the phenotype of MF9/sPrdm16 cells such as resistance to AraC and high OxPHOS status. Furthermore, 10058-F4, a c-Myc inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells. Next, we asked if the observation in MF9/sPrdm16 cells could be translated into human AML cells. Human AML cell lines, MOLM-13 and THP-1, with sPRDM16 overexpression also showed significantly higher IC 50 against AraC compared to Mock expressed cells. Moreover, RNA-seq data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial showed that AML blasts with high PRDM16 expression demonstrated a gene expression signature associated with “OxPHOS” and “Myc Targets”. In conclusion, our findings demonstrate that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via c-MYC, thereby conferres AraC resistance on AML cells. These results suggest that targeting mitochondrial respiration might be a novel treatment strategy to overcome chemoresistance in AML patients with high PRDM16 expression.

Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9

ObjectiveTo explore whether the ethanol extract of Herpetospermum caudigerum Wall (EHC), a Xizang medicinal plant traditionally used for treating liver diseases, can improve imiquimod-induced psoriasis-like skin inflammation. MethodsImmunohistochemistry and immunofluorescence staining were used to determine the effects of topical EHC use in vivo on the skin pathology of imiquimod-induced psoriasis in mice. The protein levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-17A (IL-17A) in mouse skin samples were examined using immunohistochemical staining. In vitro, IFN-γ-induced HaCaT cells with or without EHC treatment were used to evaluate the expression of keratinocyte-derived intercellular cell adhesion molecule-1 (ICAM-1) and chemokine CXC ligand 9 (CXCL9) using Western blotting and reverse transcription-quantitative polymerase chain reaction. The protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132 were utilized to validate the EHC-mediated mechanism underlying degradation of ICAM-1 and CXCL9. ResultsEHC improved inflammation in the imiquimod-induced psoriasis mouse model and reduced the levels of IFN-γ, TNF-α, and IL-17A in psoriatic lesions. Treatment with EHC also suppressed ICAM-1 and CXCL9 in epidermal keratinocytes. Further mechanistic studies revealed that EHC suppressed keratinocyte-derived ICAM-1 and CXCL9 by promoting ubiquitin–proteasome-mediated protein degradation rather than transcriptional repression. Seven primary compounds including ehletianol C, dehydrodiconiferyl alcohol, herpetrione, herpetin, herpetotriol, herpetetrone and herpetetrol were identified from the EHC using ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry. ConclusionTopical application of EHC ameliorates psoriasis-like skin symptoms and improves the inflammation at the lesion sites.Please cite this article as: Zhong Y, Zhang BW, Li JT, Zeng X, Pei JX, Zhang YM, Yang YX, Li FL, Deng Y, Zhao Q. Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9. J Integr Med. 2023; Epub ahead of print.