Critical Cellular Mechanism Research Articles

The Antinociceptive Role of Nrf2 in Neuropathic Pain: From Mechanisms to Clinical Perspectives.

Neuropathic pain, a chronic condition resulting from nerve injury or dysfunction, presents significant therapeutic challenges and is closely associated with oxidative stress and inflammation, both of which can lead to mitochondrial dysfunction. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, a critical cellular defense mechanism against oxidative stress, has emerged as a promising target for neuropathic pain management. Nrf2 modulators enhance the expression of antioxidant and cytoprotective genes, thereby reducing oxidative damage, inflammation, and mitochondrial impairment. This review explores the antinociceptive effects of Nrf2, highlighting how pharmacological agents and natural compounds may be used as potential therapeutic strategies against neuropathic pain. Although preclinical studies demonstrate significant pain reduction and improved nerve function through Nrf2 activation, several clinical challenges need to be addressed. However, emerging clinical evidence suggests potential benefits of Nrf2 modulators in several conditions, such as diabetic neuropathy and multiple sclerosis. Future research should focus on further elucidating the molecular role of Nrf2 in neuropathic pain to optimize its modulation efficacy and maximize clinical utility.

Quiescence-Origin Senescence: A New Paradigm in Cellular Aging.

Cellular senescence, traditionally viewed as a consequence of proliferating and growing cells overwhelmed by extensive stresses and damage, has long been recognized as a critical cellular aging mechanism. Recent research, however, has revealed a novel pathway termed "quiescence-origin senescence", where cells directly transition into senescence from the quiescent state, bypassing cell proliferation and growth. This opinion paper presents a framework conceptualizing a continuum between quiescence and senescence with quiescence deepening as a precursor to senescence entry. We explore the triggers and controllers of this process and discuss its biological implications. Given that the majority of cells in the human body are dormant rather than proliferative, understanding quiescence-origin senescence has significant implications for tissue homeostasis, aging, cancer, and various disease processes. The new paradigm in exploring this previously overlooked senescent cell population may reshape our intervention strategies for age-related diseases and tissue regeneration.

Failure of Autophagy in Pompe Disease.

Autophagy is an evolutionarily conserved lysosome-dependent degradation of cytoplasmic constituents. The system operates as a critical cellular pro-survival mechanism in response to nutrient deprivation and a variety of stress conditions. On top of that, autophagy is involved in maintaining cellular homeostasis through selective elimination of worn-out or damaged proteins and organelles. The autophagic pathway is largely responsible for the delivery of cytosolic glycogen to the lysosome where it is degraded to glucose via acid α-glucosidase. Although the physiological role of lysosomal glycogenolysis is not fully understood, its significance is highlighted by the manifestations of Pompe disease, which is caused by a deficiency of this lysosomal enzyme. Pompe disease is a severe lysosomal glycogen storage disorder that affects skeletal and cardiac muscles most. In this review, we discuss the basics of autophagy and describe its involvement in the pathogenesis of muscle damage in Pompe disease. Finally, we outline how autophagic pathology in the diseased muscles can be used as a tool to fast track the efficacy of therapeutic interventions.

Understanding the Unfolded Protein Response (UPR) Pathway: Insights into Neuropsychiatric Disorders and Therapeutic Potentials.

The Unfolded Protein Response (UPR) serves as a critical cellular mechanism dedicated to maintaining protein homeostasis, primarily within the endoplasmic reticulum (ER). This pathway diligently responds to a variety of intracellular indicators of ER stress with the objective of reinstating balance by diminishing the accumulation of unfolded proteins, amplifying the ER's folding capacity, and eliminating slow-folding proteins. Prolonged ER stress and UPR irregularities have been linked to a range of neuropsychiatric disorders, including major depressive disorder, bipolar disorder, and schizophrenia. This review offers a comprehensive overview of the UPR pathway, delineating its activation mechanisms and its role in the pathophysiology of neuropsychiatric disorders. It highlights the intricate interplay within the UPR and its profound influence on brain function, synaptic perturbations, and neural developmental processes. Additionally, it explores evolving therapeutic strategies targeting the UPR within the context of these disorders, underscoring the necessity for precision and further research to effective treatments. The research findings presented in this work underscore the promising potential of UPR-focused therapeutic approaches to address the complex landscape of neuropsychiatric disorders, giving rise to optimism for improving outcomes for individuals facing these complex conditions.

Epigenetic orchestration of the DNA damage response: Insights into the regulatory mechanisms.

The DNA damage response (DDR) is a critical cellular mechanism that safeguards genome integrity and prevents the accumulation of harmful DNA lesions. Increasing evidence highlights the intersection between DDR signaling and epigenetic regulation, offering profound insights into various aspects of cellular function including oncogenesis. This comprehensive review explores the intricate relationship between the epigenetic modifications and DDR activation, with a specific focus on the impact of viral infections. Oncogenic viruses, such as human papillomavirus, hepatitis virus (HBV or HCV), and Epstein-Barr virushave been shown to activate the DDR. Consequently, these DNA damage events trigger a cascade of epigenetic alterations, including changes in DNA methylation patterns, histone modifications and the expression of noncoding RNAs. These epigenetic changes exert profound effects on chromatin structure, gene expression, and maintenance of genome stability. Importantly, elucidation of the viral-induced epigenetic alterations in the context of DDR holds significant implications for comprehending the complexity of cancer and provides potential targets for therapeutic interventions.

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide’s affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway

Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

Multi-omics approaches identify SF3B3 and SIRT3 as candidate autophagic regulators and druggable targets in invasive breast carcinoma

Autophagy is a critical cellular homeostatic mechanism, and its dysfunction is linked to invasive breast carcinoma (BRCA). Recently, several omics methods have been applied to explore autophagic regulators in BRCA; however, more reliable and robust approaches for identifying crucial regulators and druggable targets remain to be discovered. Thus, we report here the results of multi-omics approaches to identify potential autophagic regulators in BRCA, including gene expression (EXP), DNA methylation (MET) and copy number alterations (CNAs) from The Cancer Genome Atlas (TCGA). Newly identified candidate genes, such as SF3B3, TRAPPC10, SIRT3, MTERFD1, and FBXO5, were confirmed to be involved in the positive or negative regulation of autophagy in BRCA. SF3B3 was identified firstly as a negative autophagic regulator, and siRNA/shRNA-SF3B3 were shown to induce autophagy-associated cell death in in vitro and in vivo breast cancer models. Moreover, a novel small-molecule activator of SIRT3, 1-methylbenzylamino amiodarone, was discovered to induce autophagy in vitro and in vivo. Together, these results provide multi-omics approaches to identify some key candidate autophagic regulators, such as the negative regulator SF3B3 and positive regulator SIRT3 in BRCA, and highlight SF3B3 and SIRT3 as new druggable targets that could be used to fill the gap between autophagy and cancer drug development.

The identification and characterisation of autophagy inhibitors from the published kinase inhibitor sets.

Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.

Targeted and Interactome Proteomics Revealed the Role of PHD2 in Regulating BRD4 Proline Hydroxylation

Proline hydroxylation is a critical cellular mechanism regulating energy homeostasis and development. Our previous study identified and validated Bromodomain-containing protein 4 (BRD4) as a proline hydroxylation substrate in cancer cells. Yet, the regulatory mechanism and the functional significance of the modification remain unknown. In this study, we developed targeted quantification assays using parallel-reaction monitoring and biochemical analysis to identify the major regulatory enzyme of BRD4 proline hydroxylation. We further performed quantitative interactome analysis to determine the functional significance of the modification pathway in BRD4-mediated protein-protein interactions and gene transcription. Our findings revealed that PHD2 is the key regulatory enzyme of BRD4 proline hydroxylation and the modification significantly affects BRD4 interactions with key transcription factors as well as BRD4-mediated transcriptional activation. Taken together, this study provided mechanistic insights into the oxygen-dependent modification of BRD4 and revealed new roles of the pathway in regulating BRD4-dependent gene expression.

A toolkit for studying cell surface shedding of diverse transmembrane receptors.

Proteolysis of transmembrane receptors is a critical cellular communication mechanism dysregulated in disease, yet decoding proteolytic regulation mechanisms of hundreds of shed receptors is hindered by difficulties controlling stimuli and unknown fates of cleavage products. Notch proteolytic regulation is a notable exception, where intercellular forces drive exposure of a cryptic protease site within a juxtamembrane proteolytic switch domain to activate transcriptional programs. We created a Synthetic Notch Assay for Proteolytic Switches (SNAPS) that exploits the modularity and unequivocal input/response of Notch proteolysis to screen surface receptors for other putative proteolytic switches. We identify several new proteolytic switches among receptors with structural homology to Notch. We demonstrate SNAPS can detect shedding in chimeras of diverse cell surface receptors, leading to new, testable hypotheses. Finally, we establish the assay can be used to measure modulation of proteolysis by potential therapeutics and offer new mechanistic insights into how DECMA-1 disrupts cell adhesion.

The Capsule of Cryptococcus neoformans Modulates Phagosomal pH through Its Acid-Base Properties.

Phagosomal acidification is a critical cellular mechanism for the inhibition and killing of ingested microbes by phagocytic cells. The acidic environment activates microbicidal proteins and creates an unfavorable environment for the growth of many microbes. Consequently, numerous pathogenic microbes have developed strategies for countering phagosomal acidification through various mechanisms that include interference with phagosome maturation. The human-pathogenic fungus Cryptococcus neoformans resides in acidic phagosomes after macrophage ingestion that actually provides a favorable environment for replication, since the fungus replicates faster at acidic pH. We hypothesized that the glucuronic acid residues in the capsular polysaccharide had the capacity to affect phagosomal acidity through their acid-base properties. A ratiometric fluorescence comparison of imaged phagosomes containing C. neoformans to phagosomes containing beads showed that the latter were significantly more acidic. Similarly, phagosomes containing nonencapsulated C. neoformans cells were more acidic than those containing encapsulated cells. Acid-base titrations of isolated C. neoformans polysaccharide revealed that it behaves as a weak acid with maximal buffering capacity around pH 4 to 5. We interpret these results as indicating that the glucuronic acid residues in the C. neoformans capsular polysaccharide can buffer phagosomal acidification. Interference with phagosomal acidification represents a new function for the cryptococcal capsule in virulence and suggests the importance of considering the acid-base properties of microbial capsules in the host-microbe interaction for other microbes with charged residues in their capsules.IMPORTANCECryptococcus neoformans is the causative agent of cryptococcosis, a devastating fungal disease that affects thousands of individuals worldwide. This fungus has the capacity to survive inside phagocytic cells, which contributes to persistence of infection and dissemination. One of the major antimicrobial mechanisms of host phagocytes is to acidify the phagosomal compartment after ingestion of microbes. This study shows that the capsule of C. neoformans can interfere with full phagosomal acidification by serving as a buffer.

Virulent Poxviruses Inhibit DNA Sensing by Preventing STING Activation.

ABSTRACTCytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerizes and translocates from the endoplasmic reticulum (ER) to a perinuclear region to mediate IRF-3 activation. Poxviruses are double-stranded DNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here, we investigated the activation of innate immune signaling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerized and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerization and phosphorylation during infection and in response to transfected DNA and cyclic GMP-AMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence.IMPORTANCE Poxviruses are double-stranded DNA viruses infecting a wide range of vertebrates and include the causative agent of smallpox (variola virus) and its vaccine vaccinia virus (VACV). Despite smallpox eradication VACV remains of interest as a therapeutic. Attenuated strains are popular vaccine candidates, whereas replication-competent strains are emerging as efficient oncolytics in virotherapy. The successful therapeutic use of VACV depends on a detailed understanding of its ability to modulate host innate immune responses. DNA sensing is a critical cellular mechanism for pathogen detection and activation of innate immunity that is centrally coordinated by the endoplasmic reticulum-resident protein STING. Here, STING is shown to mediate immune activation in response to MVA, but not in response to virulent VACV strains or other virulent poxviruses, which prevent STING activation and DNA sensing during infection and after DNA transfection. These results provide new insights into poxvirus immune evasion and have implications in the rational design of VACV-based therapeutics.

Muscarinic Receptor-Dependent Long Term Depression in the Perirhinal Cortex and Recognition Memory are Impaired in the rTg4510 Mouse Model of Tauopathy

Neurodegenerative diseases affecting cognitive dysfunction, such as Alzheimer’s disease and fronto-temporal dementia, are often associated impairments in the visual recognition memory system. Recent evidence suggests that synaptic plasticity, in particular long term depression (LTD), in the perirhinal cortex (PRh) is a critical cellular mechanism underlying recognition memory. In this study, we have examined novel object recognition and PRh LTD in rTg4510 mice, which transgenically overexpress tauP301L. We found that 8–9 month old rTg4510 mice had significant deficits in long- but not short-term novel object recognition memory. Furthermore, we also established that PRh slices prepared from rTg4510 mice, unlike those prepared from wildtype littermates, could not support a muscarinic acetylcholine receptor-dependent form of LTD, induced by a 5 Hz stimulation protocol. In contrast, bath application of the muscarinic agonist carbachol induced a form of chemical LTD in both WT and rTg4510 slices. Finally, when rTg4510 slices were preincubated with the acetylcholinesterase inhibitor donepezil, the 5 Hz stimulation protocol was capable of inducing significant levels of LTD. These data suggest that dysfunctional cholinergic innervation of the PRh of rTg4510 mice, results in deficits in synaptic LTD which may contribute to aberrant recognition memory in this rodent model of tauopathy.

Prenatal Ethanol Exposure Persistently Alters Endocannabinoid Signaling and Endocannabinoid-Mediated Excitatory Synaptic Plasticity in Ventral Tegmental Area Dopamine Neurons

Prenatal ethanol exposure (PE) leads to increased addiction risk which could be mediated by enhanced excitatory synaptic strength in ventral tegmental area (VTA) dopamine (DA) neurons. Previous studies have shown that PE enhances excitatory synaptic strength by facilitating an anti-Hebbian form of long-term potentiation (LTP). In this study, we investigated the effect of PE on endocannabinoid-mediated long-term depression (eCB-LTD) in VTA DA neurons. Rats were exposed to moderate (3 g/kg/d) or high (6 g/kg/d) levels of ethanol during gestation. Whole-cell recordings were conducted in male offspring between 4 and 10 weeks old.We found that PE led to increased amphetamine self-administration. Both moderate and high levels of PE persistently reduced low-frequency stimulation-induced eCB-LTD. Furthermore, action potential-independent glutamate release was regulated by tonic eCB signaling in PE animals. Mechanistic studies for impaired eCB-LTD revealed that PE downregulated CB1 receptor function. Interestingly, eCB-LTD in PE animals was rescued by metabotropic glutamate receptor I activation, suggesting that PE did not impair the synthesis/release of eCBs. In contrast, eCB-LTD in PE animals was not rescued by increasing presynaptic activity, which actually led to LTP in PE animals, whereas LTD was still observed in controls. This result shows that the regulation of excitatory synaptic plasticity is fundamentally altered in PE animals. Together, PE leads to impaired eCB-LTD at the excitatory synapses of VTA DA neurons primarily due to CB1 receptor downregulation. This effect could contribute to enhanced LTP and the maintenance of augmented excitatory synaptic strength in VTA DA neurons and increased addiction risk after PE.SIGNIFICANCE STATEMENT Prenatal ethanol exposure (PE) is among many adverse developmental factors known to increase drug addiction risk. Increased excitatory synaptic strength in VTA DA neurons is a critical cellular mechanism for addiction risk. Our results show that PE persistently alters eCB signaling and impairs eCB-LTD at the excitatory synapses, an important synaptic plasticity that weakens synaptic strength. These effects combined with PE-induced anti-Hebbian long-term potentiation reported in a previous study could result in the maintenance of enhanced excitatory synaptic strength in VTA DA neurons, which in turn contributes to PE-induced increase in addiction risk. Our findings also suggest that restoring normal eCB signaling in VTA DA neurons could be a useful strategy for treating behavioral symptoms caused by PE.

The role of autophagy in hepatocellular carcinoma: friend or foe.

Autophagy is an evolutionarily conserved lysosome-dependent catabolic process which degrades cell’s components in order to recycle substrates to exert optimally and adapt to tough circumstances. It is a critical cellular homeostatic mechanism with stress resistance, immunity, antiaging, and pro-tumor or anti-tumor effects. Among these, the role of autophagy in cancer is the most eye-catching that is not immutable but dynamic and highly complex. Basal autophagy acts as a tumor suppressor by maintaining genomic stability in normal cells. However, once a tumor is established, unbalanced autophagy will contribute to carcinoma cell survival under tumor microenvironment and in turn promote tumor growth and development. The dynamic role of autophagy can also apply on hepatocellular carcinoma (HCC). HCC is a highly malignant cancer with high morbidity and poor survival rate. Decline or overexpression of autophagic essential genes such as ATG7, ATG5 or Beclin 1 plays a key role in the occurrence and development of HCC but the exact mechanisms are still highly controversial. Signaling pathways or molecules involving in autophagy, for example PI3K/AKT/mTOR pathway, ERK/MAPK pathway, PERK pathway, p53, LncRNA PTENP1 (Long non-coding RNA PTENP1), microRNA-375 and so on, occupy an important position in the complex role of autophagy in HCC. Here, we discuss the dynamic role, the signaling pathways and the potential prognostic and therapy value of autophagy in HCC.

A role for autophagy in carotid atherosclerosis.

Autophagy has emerged in recent years as a critical cellular survival mechanism for cell homeostasis and may play a protective role in atherosclerosis. We aimed to review here the role autophagy plays in different cell types present in carotid atherosclerotic plaques and that may be associated with the development of unstable carotid atheroma plaque. We performed a thorough literature exploration in this area of research covering the three main cell types present in carotid atheroma plaques. Reviewed reports indicate that the role of autophagy in stable or unstable carotid atherosclerotic plaques depends on the different cell types and phenotypes, the stage and morphology of the plaque and the specific autophagy factor/s involved. Although defective autophagy could be one of the causes for carotid atheroma plaques to become unstable, it is important to take into account that autophagic players can act differentially in different cell types and different stages of the developed plaque. This review provides an overview of the role of autophagy in the main cell types in carotid atherosclerosis (i.e. macrophages, endothelial cells and smooth muscle cells).

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells.

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells.

Interactive effects of AM251 and baclofen on synaptic plasticity in the rat dentate gyrus

Long-term potentiation (LTP), a form of synaptic plasticity, is considered to be a critical cellular mechanism that underlies learning and memory. Cannabinoid CB1 and metabotropic GABAB receptors display similar pharmacological effects and co-localize in certain brain regions. In this study, we examined the effects of co-administration of the CB1 and GABAB antagonists AM251 and baclofen, respectively, on LTP induction in the rat dentate gyrus (DG).Male Wistar rats were anesthetized with urethane. A stimulating electrode was placed in the lateral perforant path (PP), and a bipolar recording electrode was inserted into the DG until maximal field excitatory postsynaptic potentials (fEPSPs) were observed. LTP was induced in the hippocampal area by high-frequency stimulation (HFS) of the PP. fEPSPs and population spikes (PS) were recorded at 5, 30, and 60min after HFS in order to measure changes in the synaptic responses of DG neurons.Our results showed that HFS coupled with administration of AM251 and baclofen increased both PS amplitude and fEPSP slope. Furthermore, co-administration of AM251 and baclofen elicited greater increases in PS amplitude and fEPSP slope.The results of the present study suggest that CB1 receptor activation in the hippocampus mainly modifies synapses onto GABAergic interneurons located in the DG. Our results further suggest that, when AM251 and baclofen are administered simultaneously, AM251 can alter GABA release and thereby augment LTP through GABAB receptors. These results suggest that functional crosstalk between cannabinoid and GABA receptors regulates hippocampal synaptic plasticity.