Role of TRPA1 in Painful Cold Hypersensitivity.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that plays a pivotal role in pain generation after exposure to irritant chemicals and is involved in the sensation of a wide variety of pathological pain. TRPA1 was first reported to be sensitive to noxious cold, but its intrinsic cold sensitivity still remains under debate. To address this issue, we focused on cold hypersensitivity induced by oxaliplatin, a platinum-based chemotherapeutic drug, as a peculiar adverse symptom of acute peripheral neuropathy. We and other groups have shown that oxaliplatin enhances TRPA1 sensitivity to its chemical agonists and reactive oxygen species (ROS). Our in vitro and animal model studies revealed that oxaliplatin, or its metabolite oxalate, inhibits hydroxylation of a proline residue within the N-terminus of human TRPA1 (hTRPA1) via inhibition of prolyl hydroxylase domain-containing protein (PHD), which induces TRPA1 sensitization to ROS. Although hTRPA1 is insensitive to cold, PHD inhibition endows hTRPA1 with cold sensitivity through sensing the small amount of ROS produced after exposure to cold. Hence, we propose that PHD inhibition can unveil the cold sensitivity of hTRPA1 by converting ROS signaling into cold sensitivity. Furthermore, in this review, we summarize the role of TRPA1 in painful cold hypersensitivity during peripheral vascular impairment.

The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex.

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Redox Enzymes P4HB and PDIA3 Interact with STIM1 to Fine-Tune Its Calcium Sensitivity and Activation.

Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.

P4HA2 promotes proliferation, invasion, and metastasis through regulation of the PI3K/AKT signaling pathway in oral squamous cell carcinoma.

Proline 4-hydroxylase 2 (P4HA2) is known for its hydroxylase activity, primarily involved in hydroxylating collagen precursors and promoting collagen cross-linking under physiological conditions. Although its overexpression influences a wide variety of malignant tumors' occurrence and development, its specific effects and mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. This study focused on investigating the expression patterns, carcinogenic functions, and underlying mechanisms of P4HA2 in OSCC cells. Various databases, including TCGA, TIMER, UALCAN, GEPIA, and K-M plotter, along with paraffin-embedded samples, were used to ascertain P4HA2 expression in cancer and its correlation with clinicopathological features. P4HA2 knockdown and overexpression cell models were developed to assess its oncogenic roles and mechanisms. The results indicated that P4HA2 was overexpressed in OSCC and inversely correlated with patient survival. Knockdown of P4HA2 suppressed invasion, migration, and proliferation of OSCC cells both in vitro and in vivo, whereas overexpression of P4HA2 had the opposite effects. Mechanistically, the phosphorylation levels of the PI3K/AKT pathway were reduced following P4HA2 silencing. The study reveals that P4HA2 acts as a promising biomarker for predicting prognosis in OSCC and significantly affects metastasis, invasion, and proliferation of OSCC cells through the regulation of the PI3K/AKT signaling pathway.

P4HA3 promotes head and neck squamous cell carcinoma progression via the WNT/ß-catenin signaling pathway.

Here, we explored the role of Prolyl 4-Hydroxylase Subunit Alpha 3 (P4HA3), the most recently identified member of the prolyl-4-hydroxylase (P4H) family, in head and neck squamous cell carcinoma (HNSCC) progression. P4HA3 is upregulated during cancer progression; however, its specific role in HNSCC progression remains elusive. Thus, this study aimed to elucidate the regulatory function of P4HA3 in HNSCC development and progression and to describe the underlying mechanisms. Initially, we analyzed the correlation between the expression of P4HA3 and the WNT pathway genes and clinicopathologic features in HNSCC based on microarray data from The Cancer Genome Atlas (TCGA). Next, we used Gene Oncology (GO) functional data to describe several potentially associated pathways in HNSCC. Then, we knocked down P4HA3 in SCC15 and SCC25 cells, two classic HNSCC cell lines, and assessed the resulting changes using RT-qPCR. Furthermore, we used Western blot to evaluate the regulatory role of P4HA3 in the epithelial-to-mesenchymal transition (EMT) and the WNT/ß-catenin signaling pathway. To explore the effect of P4HA3 knockdown on tumor progression, in vivo experiments were conducted using a murine model. Immunohistochemistry assays were then employed to identify proteins associated with EMT and the WNT/ß-catenin signaling pathway in tumor tissues. Upregulated P4HA3 in HNSCC patient tumor tissues was positively correlated with poor prognosis. Notably, P4HA3 knockdown significantly inhibited the proliferative and invasive abilities of HNSCC. The levels of genes and proteins associated with EMT and the WNT/ß-catenin signaling pathway were also markedly reduced by P4HA3 knockdown. Importantly, the in vivo experiments demonstrated that P4HA3 can promote subcutaneous tumorigenesis in nude mice and knockdown of P4HA3 induce a significant ihibitation of EMT and WNT/ß-catenin pathway detected by immunohistochemistry assay in tumor tissues. In summary, we demonstrated that P4HA3 is a promising diagnostic and therapeutic biomarker for HNSCC. As an oncogene, P4HA3 increases HNSCC proliferation by inducing the EMT and activating the WNT/ß-catenin signaling pathway.

Heat shock protein 90 and prolyl hydroxylase 2 co-regulate hypoxia-inducible factor-1α expression in porcine small intestinal epithelial cells under heat stress.

Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature-humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.

Icariin upregulates methyltransferase-like 14-mediated prolyl 4-hydroxylase beta subunit m6A modification to promote osteogenic differentiation of bone marrow stem cells.

Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.

P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

Comprehensive analysis revealed P4Hs as new biomarkers for prognosis and immunotherapy in head and neck cancer.

Prolyl 4-hydroxylases (P4Hs) are a family of key modifying enzymes in collagen synthesis. P4Hs have been confirmed to be closely associated with tumor occurrence and development. However, the expression of P4Hs in head and neck cancer (HNSC) as well as its relationship with prognosis and tumor immunity infiltration has not yet been analyzed. We investigated the transcriptional expression, survival data, and immune infiltration of P4Hs in patients with HNSC from multiple databases. P4H1-3 expression was significantly higher in HNSC tumor tissues than in normal tissues. Moreover, P4HA1 and P4HA2 were associated with tumor stage, patient prognosis, and immune cell infiltration. P4HA3 was related to patient prognosis and immune cell infiltration. Correlation experiments confirmed that P4HA1 may serve as a prognosis biomarker and plays a role in the progression of nasopharyngeal carcinoma. These findings suggest that P4HA1-3 may be a novel biomarker for the prognosis and treatment of HNSC, which is expected to support the development of new therapies for patients with head and neck tumors and improve patient outcomes.

Activation of the hypoxia-inducible factor pathway protects against acute ischemic stroke by reprogramming central carbon metabolism.

Cell metabolism reprogramming to sustain energy production, while reducing oxygen and energy consuming processes is crucially important for the adaptation to hypoxia/ischemia. Adaptive metabolic rewiring is controlled by hypoxia-inducible factors (HIFs). Accumulating experimental evidence indicates that timely activation of HIF in brain-resident cells improves the outcome from acute ischemic stroke. However, the underlying molecular mechanisms are still incompletely understood. Thus, we investigated whether HIF-dependent metabolic reprogramming affects the vulnerability of brain-resident cells towards ischemic stress. Methods: We used genetic and pharmacological approaches to activate HIF in the murine brain in vivo and in primary neurons and astrocytes in vitro. Numerous metabolomic approaches and molecular biological techniques were applied to elucidate potential HIF-dependent effects on the central carbon metabolism of brain cells. In animal and cell models of ischemic stroke, we analysed whether HIF-dependent metabolic reprogramming influences the susceptibility to ischemic injury. Results: Neuron-specific gene ablation of prolyl-4-hydroxylase domain 2 (PHD2) protein, negatively regulating the protein stability of HIF-α in an oxygen dependent manner, reduced brain injury and functional impairment of mice after acute stroke in a HIF-dependent manner. Accordingly, PHD2 deficient neurons showed an improved tolerance towards ischemic stress in vitro, which was accompanied by enhanced HIF-1-mediated glycolytic lactate production through pyruvate dehydrogenase kinase-mediated inhibition of the pyruvate dehydrogenase. Systemic treatment of mice with roxadustat, a low-molecular weight pan-PHD inhibitor, not only increased the abundance of numerous metabolites of the central carbon and amino acid metabolism in murine brain, but also ameliorated cerebral tissue damage and sensorimotor dysfunction after acute ischemic stroke. In neurons and astrocytes roxadustat provoked a HIF-1-dependent glucose metabolism reprogramming including elevation of glucose uptake, glycogen synthesis, glycolytic capacity, lactate production and lactate release, which enhanced the ischemic tolerance of astrocytes, but not neurons. We found that strong activation of HIF-1 in neurons by non-selective inhibition of all PHD isoenzymes caused a HIF-1-dependent upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 redirecting glucose-6-phosphate from pentose phosphate pathway (PPP) to the glycolysis pathway. This was accompanied by a reduction of NADPH production in the PPP, which further decreased the low intrinsic antioxidant reserve of neurons, making them more susceptible to ischemic stress. Nonetheless, in organotypic hippocampal cultures with preserved neuronal-glial interactions roxadustat decreased the neuronal susceptibility to ischemic stress, which was largely prevented by restricting glycolytic energy production through lactate transport blockade. Conclusion: Collectively, our results indicate that HIF-1-mediated metabolic reprogramming alleviates the intrinsic vulnerability of brain-resident cells to ischemic stress.

P4HA2 activates mTOR via hydroxylation and targeting P4HA2-mTOR inhibits lung adenocarcinoma cell growth.

Mammalian target of rapamycin (mTOR) kinase functions as a central regulator of cell growth and metabolism, and its complexes mTORC1 and mTORC2 phosphorylate distinct substrates. Dysregulation of mTOR signaling is commonly implicated in human diseases, including cancer. Despite three decades of active research in mTOR, much remains to be determined. Here, we demonstrate that prolyl 4-hydroxylase alpha-2 (P4HA2) binds directly to mTOR and hydroxylates one highly conserved proline 2341 (P2341) within a kinase domain of mTOR, thereby activating mTOR kinase and downstream effector proteins (e.g. S6K and AKT). Moreover, the hydroxylation of P2341 strengthens mTOR stability and allows mTOR to accurately recognize its substrates such as S6K and AKT. The growth of lung adenocarcinoma cells overexpressing mTORP2341A is significantly reduced when compared with that of cells overexpressing mTORWT. Interestingly, in vivo cell growth assays show that targeting P4HA2-mTOR significantly suppresses lung adenocarcinoma cell growth. In summary, our study reveals an undiscovered hydroxylation-regulatory mechanism by which P4HA2 directly activates mTOR kinase, providing insights for therapeutically targeting mTOR kinase-driven cancers.

Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices.

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

PHD1-3 oxygen sensors in vivo-lessons learned from gene deletions.

Oxygen sensors enable cells to adapt to limited oxygen availability (hypoxia), affecting various cellular and tissue responses. Prolyl-4-hydroxylase domain 1-3 (PHD1-3; also called Egln1-3, HIF-P4H 1-3, HIF-PH 1-3) proteins belong to the Fe2+- and 2-oxoglutarate-dependent dioxygenase superfamily and utilise molecular oxygen (O2) alongside 2-oxoglutarate as co-substrate to hydroxylate two proline residues of α subunits of the dimeric hypoxia inducible factor (HIF) transcription factor. PHD1-3-mediated hydroxylation of HIF-α leads to its degradation and inactivation. Recently, various PHD inhibitors (PHI) have entered the clinics for treatment of renal anaemia. Pre-clinical analyses indicate that PHI treatment may also be beneficial in numerous other hypoxia-associated diseases. Nonetheless, the underlying molecular mechanisms of the observed protective effects of PHIs are only partly understood, currently hindering their translation into the clinics. Moreover, the PHI-mediated increase of Epo levels is not beneficial in all hypoxia-associated diseases and PHD-selective inhibition may be advantageous. Here, we summarise the current knowledge about the relevance and function of each of the three PHD isoforms in vivo, based on the deletion or RNA interference-mediated knockdown of each single corresponding gene in rodents. This information is crucial for our understanding of the physiological relevance and function of the PHDs as well as for elucidating their individual impact on hypoxia-associated diseases. Furthermore, this knowledge highlights which diseases may best be targeted by PHD isoform-selective inhibitors in case such pharmacologic substances become available.

HIF1α/ATF3 partake in PGK1 K191/K192 succinylation by modulating P4HA1/succinate signaling in glioblastoma.

BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSIONS: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.

Exploring the Influence of Zinc Ions on the Conformational Stability and Activity of Protein Disulfide Isomerase.

The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Hypoxia-induced P4HA1 overexpression promotes post-ischemic angiogenesis by enhancing endothelial glycolysis through downregulating FBP1.

BACKGROUND: Angiogenesis is essential for tissue repair in ischemic diseases, relying on glycolysis as its primary energy source. Prolyl 4-hydroxylase subunit alpha 1 (P4HA1), the catalytic subunit of collagen prolyl 4-hydroxylase, is a glycolysis-related gene in cancers. However, its role in glycolysis-induced angiogenesis remains unclear. METHODS: P4HA1 expression was modulated using adenoviruses. Endothelial angiogenesis was evaluated through 5-ethynyl-2'-deoxyuridine incorporation, transwell migration, and tube formation assays in vitro. In vivo experiments measured blood flow and capillary density in the hindlimb ischemia (HLI) model. Glycolytic stress assays, glucose uptake, lactate production, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were employed to assess glycolytic capacity. Transcriptome sequencing, validated by western blotting and RT-PCR, was utilized to determine underlying mechanisms. RESULTS: P4HA1 was upregulated in endothelial cells under hypoxia and in the HLI model. P4HA1 overexpression promoted angiogenesis in vitro and in vivo, while its knockdown had the opposite effect. P4HA1 overexpression reduced cellular α-ketoglutarate (α-KG) levels by consuming α-KG during collagen hydroxylation. Downregulation of α-KG reduced the protein level of a DNA dioxygenase, ten-eleven translocation 2 (TET2), and its recruitment to the fructose-1,6-biphosphatase (FBP1) promoter, resulting in decreased FBP1 expression. The decrease in FBP1 enhanced glycolytic metabolism, thereby promoting endothelial angiogenesis. CONCLUSIONS: Hypoxia-induced endothelial P4HA1 overexpression enhanced angiogenesis by promoting glycolytic metabolism reprogramming through the P4HA1/α-KG/TET2/FBP1 pathway. The study's findings underscore the significance of P4HA1 in post-ischemic angiogenesis, suggesting its therapeutic potential for post-ischemic tissue repair.

Multilevel chitosan-gelatin particles loaded with P4HA1 siRNA suppress glioma development.

It has been reported that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) promoted tumor growth and metastasis of glioma; thus, targeting P4HA1 may be a promising therapeutic strategy against glioma. In consideration of the instability of siRNA in vivo, the chitosan-gelatin microspheres loaded with P4HA1 siRNA (P4HA1 siRNA@CGM) were employed. Firstly, the gel electrophoresis and hemolytic test were performed to assess the stability and blood compatibility of P4HA1 siRNA@CGM. Then, methyl thiazolyl tetrazolium (MTT), cell colony formation, Transwell assay, wound healing assay, gliosphere formation, tube formation, and Western blot were performed to assess the effects of P4HA1 siRNA@CGM on the biological functions of glioma. Finally, 125I-labeled P4HA1 siRNA@CGM was injected into the xenograft mice, radionuclide imaging was recorded, Ki67 and terminal deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining was performed to assess the effects of P4HA1 siRNA@CGM on tumor growth and apoptosis of glioma in vivo. The results showed that P4HA1 siRNA and P4HA1 siRNA@CGM not only markedly inhibited the proliferation, metastasis, gliosphere formation, and the protein levels of interstitial markers (N-cadherin and vimentin) and the transcription factors of epithelial-mesenchymal transition (EMT) (Snail, Slug, and Twist1) in glioma cells, but also inhibited the tube formation in human brain microvascular endothelial cells (HBMECs), and P4HA1 siRNA@CGM exhibited the better inhibitory effects than P4HA1 siRNA. Above results suggested the feasibility of P4HA1 siRNA@CGM in the clinical treatment of glioma.

Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

Prolyl hydroxylase 2 inhibits glycolytic activity in colorectal cancer via the NF­κB signaling pathway.

A variety of malignancies preferentially meet energy demands through the glycolytic pathway. Hypoxia­induced cancer cell adaptations are essential for tumor development. However, in cancerous glycolysis, the functional importance and underlying molecular mechanism of prolyl hydroxylase domain protein 2 (PHD2) have not been fully elucidated. Gain­ and loss­of­function assays were conducted to evaluate PHD2 functions in colon cancer cells. Glucose uptake, lactate production and intracellular adenosine­5'­triphosphate/adenosine diphosphate ratio were measured to determine glycolytic activities. Protein and gene expression levels were measured by western blot analysis and reverse transcription­quantitative PCR, respectively. The human colon cancer xenograft model was used to confirm the role of PHD2 in tumor progression in vivo. Functionally, the data demonstrated that PHD2 knockdown leads to increased glycolysis, while PHD2 overexpression resulted in suppressed glycolysis in colorectal cancer cells. In addition, the glycolytic activity was enhanced without PHD2 and normalized after PHD2 reconstitution. PHD2 was shown to inhibit colorectal tumor growth, suppress cancer cell proliferation and improve tumor­bearing mice survival in vivo. Mechanically, it was found that PHD2 inhibits the expression of critical glycolytic enzymes (glucose transporter 1, hexokinase 2 and phosphoinositide­dependent protein kinase 1). In addition, PHD2 inhibited Ikkß­mediated NF­κB activation in a hypoxia­inducible factor­1α­independent manner. In conclusion, the data demonstrated that PHD2/Ikkß/NF­κB signaling has critical roles in regulating glycolysis and suggests that PHD2 potentially suppresses colorectal cancer.