Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

BACKGROUND: An intergenic region at chromosome 4q31 is one of the most significant regions associated with COPD susceptibility and lung function in GWAS. In this region, the implicated causal gene HHIP has a unique epithelial expression pattern in adult human lungs, in contrast to dominant expression in fibroblasts in murine lungs. However, the mechanism underlying the species-dependent cell type-specific regulation of HHIP remains largely unknown. METHODS: We employed snATAC-seq analysis to identify open chromatin regions within the COPD GWAS region in various human lung cell types. ChIP-quantitative PCR, reporter assays, chromatin conformation capture assays and Hi-C assays were conducted to characterize the regulatory element in this region. CRISPR/Cas9-editing was performed in BEAS-2B cells to generate single colonies with stable knockout of the regulatory element. RT-PCR and Western blot assays were used to evaluate expression of HHIP and epithelial-mesenchymal transition (EMT)-related marker genes. FINDINGS: We identified a distal enhancer within the COPD 4q31 GWAS locus that regulates HHIP transcription at baseline and after TGFß treatment in a SMAD3-dependent, but Hedgehog-independent manner in human bronchial epithelial cells. The distal enhancer also maintains chromatin topological domains near 4q31 locus and HHIP gene. Reduced HHIP expression led to increased EMT induced by TGFß in human bronchial epithelial cells. INTERPRETATION: A distal enhancer regulates HHIP expression both under homeostatic condition and upon TGFß treatment in human bronchial epithelial cells. The interaction between HHIP and TGFß signalling possibly contributes to COPD pathogenesis. FUNDING: Supported by NIH grants R01HL127200, R01HL148667 and R01HL162783 (to X. Z).

Rapid Antigen Diagnostics as Frontline Testing in the COVID-19 Pandemic.

The ongoing global COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in significant loss of life since December 2019. Timely and precise virus detection has been proven as an effective solution to reduce the spread of the virus and to track the epidemic. Rapid antigen diagnostics has played a significant role in the frontline of COVID-19 testing because of its convenience, low cost, and high accuracy. Herein, different types of recently innovated in-lab and commercial antigen diagnostic technologies with emphasis on the strengths and limitations of these technologies including the limit of detection, sensitivity, specificity, affordability, and usability are systematically reviewed. The perspectives of assay development are looked into.

SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

Deficiency in the clearance of cellular debris is a major pathogenic factor in the emergence of autoimmune diseases. We previously demonstrated that mice deficient for scavenger receptor class F member 1 (SCARF1) develop a lupus-like autoimmune disease with symptoms similar to human systemic lupus erythematosus (SLE), including a pronounced accumulation of apoptotic cells (ACs). Therefore, we hypothesized that SCARF1 will be important for clearance of ACs and maintenance of self-tolerance in humans, and that dysregulation of this process could contribute to SLE. In this article, we show that SCARF1 is highly expressed on phagocytic cells, where it functions as an efferocytosis receptor. In healthy individuals, we discovered that engagement of SCARF1 by ACs on BDCA1+ dendritic cells initiates an IL-10 anti-inflammatory response mediated by the phosphorylation of STAT1 and STAT3. Unexpectedly, there was no significant difference in SCARF1 expression in samples of patients with SLE compared with healthy donor samples. However, we detected anti-SCARF1 autoantibodies in 26% of patients with SLE, which was associated with dsDNA Ab positivity. Furthermore, our data show a direct correlation of the levels of anti-SCARF1 in the serum and defects in the removal of ACs. Depletion of Ig restores efferocytosis in SLE serum, suggesting that defects in the removal of ACs are partially mediated by SCARF1 pathogenic autoantibodies. Our data demonstrate that human SCARF1 is an AC receptor in dendritic cells and plays a role in maintaining tolerance and homeostasis.

Handheld Microfluidic Filtration Platform Enables Rapid, Low-Cost, and Robust Self-Testing of SARS-CoV-2 Virus.

Here, a novel microfluidic test kit combining ultrahigh throughput hydrodynamic filtration and sandwich immunoassay is reported. Specifically, nano and microbeads coated with two different, noncompetitive antibodies, are used to capture the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) proteins simultaneously, forming larger complexes. Microfluidic filtration discards free nanobeads but retains antigen-bridged complexes in the observation zone, where a display of red color indicates the presence of antigen in the sample. This testing platform exhibits high throughput separation (<30 s) and enrichment of antigen that exceeds the traditional lateral flow assays or microfluidic assays, with a low limit of detection (LoD) < 100 copies mL-1 . In two rounds of clinical trials conducted in December 2020 and August 2021, the assays demonstrate high sensitivities of 95.4% and 100%, respectively, which proves this microfluidic test kit is capable of detecting SARS-CoV-2 virus variants evolved over significant periods of time. Furthermore, the mass-produced chip can be fabricated at a cost of $0.98/test and the robust design allows the chip to be reused for over 50 times. All of these features make the microfluidic test kit particularly suitable for areas with inadequate medical infrastructure and a shortage of laboratory resources.

Hedgehog interacting protein (HHIP) represses airway remodeling and metabolic reprogramming in COPD-derived airway smooth muscle cells.

Although HHIP locus has been consistently associated with the susceptibility to COPD including airway remodeling and emphysema in genome-wide association studies, the molecular mechanism underlying this genetic association remains incompletely understood. By utilizing Hhip+/- mice and primary human airway smooth muscle cells (ASMCs), here we aim to determine whether HHIP haploinsufficiency increases airway smooth muscle mass by reprogramming glucose metabolism, thus contributing to airway remodeling in COPD pathogenesis. The mRNA levels of HHIP were compared in normal and COPD-derived ASMCs. Mitochondrial oxygen consumption rate and lactate levels in the medium were measured in COPD-derived ASMCs with or without HHIP overexpression as readouts of glucose oxidative phosphorylation and aerobic glycolysis rates. The proliferation rate was measured in healthy and COPD-derived ASMCs treated with or without 2-DG. Smooth muscle mass around airways was measured by immunofluorescence staining for α-smooth muscle actin (α-SMA) in lung sections from Hhip+/- mice and their wild type littermates, Hhip+/+ mice. Airway remodeling was assessed in Hhip+/- and Hhip+/- mice exposed to 6 months of cigarette smoke. Our results show HHIP inhibited aerobic glycolysis and represses cell proliferation in COPD-derived ASMCs. Notably, knockdown of HHIP in normal ASMCs increased PKM2 activity. Importantly, Hhip+/- mice demonstrated increased airway remodeling and increased intensity of α-SMA staining around airways compared to Hhip+/+ mice. In conclusion, our findings suggest that HHIP represses aerobic glycolysis and ASMCs hyperplasia, which may contribute to the increased airway remodeling in Hhip+/- mice.

Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling.

O-GlcNAcylation is a reversible serine/threonine glycosylation on cytosolic and nuclear proteins that are involved in various regulatory pathways. However, the detection and quantification of O-GlcNAcylation substrates have been challenging. Here, we report a highly efficient method for the identification of O-GlcNAc modification via tandem glycan labeling, in which O-GlcNAc is first galactosylated and then sialylated with a fluorophore-conjugated sialic acid residue, therefore enabling highly sensitive fluorescent detection. The method is validated on various proteins that are known to be modified by O-GlcNAcylation including CK2, NOD2, SREBP1c, AKT1, PKM, and PFKFB3, and on the nuclear extract of HEK293 cells. Using this method, we then report the evidence that hypoxia-inducible factor HIF1α is a potential target for O-GlcNAcylation, suggesting a possibly direct connection between the metabolic O-GlcNAc pathway and the hypoxia pathway.

Roles of Microglial and Monocyte Chemokines and Their Receptors in Regulating Alzheimer's Disease-Associated Amyloid-ß and Tau Pathologies.

Chemokines and their receptors have been shown to affect amyloid-ß (Aß) and tau pathologies in mouse models of Alzheimer's disease (AD) by regulating microglia and monocyte-associated neuroinflammation, microglial movement and monocyte recruitment into the brain. These cells in turn can promote and mediate Aß phagocytosis and degradation and tau phosphorylation. In this review we discuss published work in this field in mouse models of AD and review what is known about the contributions of microglial and monocyte chemokines and their receptors to amyloid and tau pathologies. We focus on the roles of the chemokine/chemokine receptor pairs CCL2/CCR2, CX3CL1/CX3CR1, CCL5/CCR5, CXCL10/CXCR3 and CXCL1/CXCR2, highlighting important knowledge gaps in this field. A full understanding of the functions of chemokines and their receptors in AD may guide the development of novel immunotherapies for this devastating disease.

Metabolomic profiling in a Hedgehog Interacting Protein (Hhip) murine model of chronic obstructive pulmonary disease.

Genetic variants annotated to the hedgehog interacting protein (HHIP) are robustly associated with chronic obstructive pulmonary disease (COPD). Hhip haploinsufficiency in mice leads to increased susceptibility towards the development of emphysema following exposure to chronic cigarette smoke (CS). To explore the molecular pathways which contribute to increased susceptibility, we performed metabolomic profiling using high performance liquid chromatography tandem mass spectroscopy (LC/MS-MS) on plasma, urine, and lung tissue of Hhip +/- heterozygotes and wild type (Hhip +/+) C57/BL6 mice exposed to either room-air or CS for six months. Univariate comparisons between groups were made with a combined fold change ≥2 and Student's t-test p-value < 0.05 to denote significance; associations with mean alveolar chord length (MACL), a quantitative measure of emphysema, and gene-by-environment interactions were examined using empiric Bayes-mediated linear models. Decreased urinary excretion of cotinine despite comparable plasma levels was observed in Hhip +/- heterozygotes; a strong gene-by-smoking association was also observed. Correlations between MACL and markers of oxidative stress such as urinary methionine sulfoxide were observed in Hhip +/- but not in Hhip +/+ mice. Metabolite set enrichment analyses suggest reduced antioxidant capacity and alterations in macronutrient metabolism contribute to increased susceptibility to chronic CS-induced oxidative stress in Hhip haploinsufficiency states.

Transcriptomic Analysis of Lung Tissue from Cigarette Smoke-Induced Emphysema Murine Models and Human Chronic Obstructive Pulmonary Disease Show Shared and Distinct Pathways.

Although cigarette smoke (CS) is the primary risk factor for chronic obstructive pulmonary disease (COPD), the underlying molecular mechanisms for the significant variability in developing COPD in response to CS are incompletely understood. We performed lung gene expression profiling of two different wild-type murine strains (C57BL/6 and NZW/LacJ) and two genetic models with mutations in COPD genome-wide association study genes (HHIP and FAM13A) after 6 months of chronic CS exposure and compared the results to human COPD lung tissues. We identified gene expression patterns that correlate with severity of emphysema in murine and human lungs. Xenobiotic metabolism and nuclear erythroid 2-related factor 2-mediated oxidative stress response were commonly regulated molecular response patterns in C57BL/6, Hhip+/-, and Fam13a-/- murine strains exposed chronically to CS. The CS-resistant Fam13a-/- mouse and NZW/LacJ strain revealed gene expression response pattern differences. The Fam13a-/- strain diverged in gene expression compared with C57BL/6 control only after CS exposure. However, the NZW/LacJ strain had a unique baseline expression pattern, enriched for nuclear erythroid 2-related factor 2-mediated oxidative stress response and xenobiotic metabolism, and converged to a gene expression pattern similar to the more susceptible wild-type C57BL/6 after CS exposure. These results suggest that distinct molecular pathways may account for resistance to emphysema. Surprisingly, there were few genes commonly modulated in mice and humans. Our study suggests that gene expression responses to CS may be largely species and model dependent, yet shared pathways could provide biologically significant insights underlying individual susceptibility to CS.

Functional interactors of three genome-wide association study genes are differentially expressed in severe chronic obstructive pulmonary disease lung tissue.

In comparison to genome-wide association studies (GWAS), there has been poor replication of gene expression studies in chronic obstructive pulmonary disease (COPD). We performed microarray gene expression profiling on a large sample of resected lung tissues from subjects with severe COPD. Comparing 111 COPD cases and 40 control smokers, 204 genes were differentially expressed; none were at significant GWAS loci. The top differentially expressed gene was HMGB1, which interacts with AGER, a known COPD GWAS gene. Differentially expressed genes showed enrichment for putative interactors of the first three identified COPD GWAS genes IREB2, HHIP, and FAM13A, based on gene sets derived from protein and RNA binding studies, RNA-interference, a murine smoking model, and expression quantitative trait locus analyses. The gene module most highly associated for COPD in Weighted Gene Co-Expression Network Analysis (WGCNA) was enriched for B cell pathways, and shared seventeen genes with a mouse smoking model and twenty genes with previous emphysema studies. As in other common diseases, genes at COPD GWAS loci were not differentially expressed; however, using a combination of network methods, experimental studies and careful phenotype definition, we found differential expression of putative interactors of these genes, and we replicated previous human and mouse microarray results.

Hhip haploinsufficiency sensitizes mice to age-related emphysema.

Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip(+/-)), we observed increased lung compliance and spontaneous emphysema in Hhip(+/-) mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip(+/-) vs. Hhip(+/+) mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip(+/-) mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip(+/-) mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip(+/-) mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.

A Chronic Obstructive Pulmonary Disease Susceptibility Gene, FAM13A, Regulates Protein Stability of ß-Catenin.

RATIONALE: A genetic locus within the FAM13A gene has been consistently associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies. However, the mechanisms by which FAM13A contributes to COPD susceptibility are unknown. OBJECTIVES: To determine the biologic function of FAM13A in human COPD and murine COPD models and discover the molecular mechanism by which FAM13A influences COPD susceptibility. METHODS: Fam13a null mice (Fam13a(-/-)) were generated and exposed to cigarette smoke. The lung inflammatory response and airspace size were assessed in Fam13a(-/-) and Fam13a(+/+) littermate control mice. Cellular localization of FAM13A protein and mRNA levels of FAM13A in COPD lungs were assessed using immunofluorescence, Western blotting, and reverse transcriptase-polymerase chain reaction, respectively. Immunoprecipitation followed by mass spectrometry identified cellular proteins that interact with FAM13A to reveal insights on FAM13A's function. MEASUREMENTS AND MAIN RESULTS: In murine and human lungs, FAM13A is expressed in airway and alveolar type II epithelial cells and macrophages. Fam13a null mice (Fam13a(-/-)) were resistant to chronic cigarette smoke-induced emphysema compared with Fam13a(+/+) mice. In vitro, FAM13A interacts with protein phosphatase 2A and recruits protein phosphatase 2A with glycogen synthase kinase 3ß and ß-catenin, inducing ß-catenin degradation. Fam13a(-/-) mice were also resistant to elastase-induced emphysema, and this resistance was reversed by coadministration of a ß-catenin inhibitor, suggesting that FAM13A could increase the susceptibility of mice to emphysema development by inhibiting ß-catenin signaling. Moreover, human COPD lungs had decreased protein levels of ß-catenin and increased protein levels of FAM13A. CONCLUSIONS: We show that FAM13A may influence COPD susceptibility by promoting ß-catenin degradation.

Exome Array Analysis Identifies a Common Variant in IL27 Associated with Chronic Obstructive Pulmonary Disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD) susceptibility is in part related to genetic variants. Most genetic studies have been focused on genome-wide common variants without a specific focus on coding variants, but common and rare coding variants may also affect COPD susceptibility. OBJECTIVES: To identify coding variants associated with COPD. METHODS: We tested nonsynonymous, splice, and stop variants derived from the Illumina HumanExome array for association with COPD in five study populations enriched for COPD. We evaluated single variants with a minor allele frequency greater than 0.5% using logistic regression. Results were combined using a fixed effects meta-analysis. We replicated novel single-variant associations in three additional COPD cohorts. MEASUREMENTS AND MAIN RESULTS: We included 6,004 control subjects and 6,161 COPD cases across five cohorts for analysis. Our top result was rs16969968 (P = 1.7 × 10(-14)) in CHRNA5, a locus previously associated with COPD susceptibility and nicotine dependence. Additional top results were found in AGER, MMP3, and SERPINA1. A nonsynonymous variant, rs181206, in IL27 (P = 4.7 × 10(-6)) was just below the level of exome-wide significance but attained exome-wide significance (P = 5.7 × 10(-8)) when combined with results from other cohorts. Gene expression datasets revealed an association of rs181206 and the surrounding locus with expression of multiple genes; several were differentially expressed in COPD lung tissue, including TUFM. CONCLUSIONS: In an exome array analysis of COPD, we identified nonsynonymous variants at previously described loci and a novel exome-wide significant variant in IL27. This variant is at a locus previously described in genome-wide associations with diabetes, inflammatory bowel disease, and obesity and appears to affect genes potentially related to COPD pathogenesis.

AMPK-HDAC5 pathway facilitates nuclear accumulation of HIF-1α and functional activation of HIF-1 by deacetylating Hsp70 in the cytosol.

Hypoxia-inducible factor 1 (HIF-1) transcriptionally promotes production of adenosine triphosphate (ATP) whereas AMPK senses and regulates cellular energy homeostasis. A histone deacetylase (HDAC) activity has been proven to be critical for HIF-1 activation but the underlying mechanism and its role in energy homesostasis remain unclear. Here, we demonstrate that HIF-1 activation depends on a cytosolic, enzymatically active HDAC5. HDAC5 knockdown impairs hypoxia-induced HIF-1α accumulation and HIF-1 transactivation, whereas HDAC5 overexpression enhances HIF-1α stabilization and nuclear translocation. Mechanistically, we show that Hsp70 is a cytosolic substrate of HDAC5; and hyperacetylation renders Hsp70 higher affinity for HIF-1α binding, which correlates with accelerated degradation and attenuated nuclear accumulation of HIF-1α. Physiologically, AMPK-triggered cytosolic shuttling of HDAC5 is critical; inhibition of either AMPK or HDAC5 impairs HIF-1α nuclear accumulation under hypoxia or low glucose conditions. Finally, we show specifically suppressing HDAC5 is sufficient to inhibit tumor cell proliferation under hypoxic conditions. Our data delineate a novel link between AMPK, the energy sensor, and HIF-1, the major driver of ATP production, indicating that specifically inhibiting HDAC5 may selectively suppress the survival and proliferation of hypoxic tumor cells.

Haploinsufficiency of Hedgehog interacting protein causes increased emphysema induced by cigarette smoke through network rewiring.

BACKGROUND: The HHIP gene, encoding Hedgehog interacting protein, has been implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS), and our subsequent studies identified a functional upstream genetic variant that decreased HHIP transcription. However, little is known about how HHIP contributes to COPD pathogenesis. METHODS: We exposed Hhip haploinsufficient mice (Hhip (+/-) ) to cigarette smoke (CS) for 6 months to model the biological consequences caused by CS in human COPD risk-allele carriers at the HHIP locus. Gene expression profiling in murine lungs was performed followed by an integrative network inference analysis, PANDA (Passing Attributes between Networks for Data Assimilation) analysis. RESULTS: We detected more severe airspace enlargement in Hhip (+/-) mice vs. wild-type littermates (Hhip (+/+) ) exposed to CS. Gene expression profiling in murine lungs suggested enhanced lymphocyte activation pathways in CS-exposed Hhip (+/-) vs. Hhip (+/+) mice, which was supported by increased numbers of lymphoid aggregates and enhanced activation of CD8+ T cells after CS-exposure in the lungs of Hhip (+/-) mice compared to Hhip (+/+) mice. Mechanistically, results from PANDA network analysis suggested a rewired and dampened Klf4 signaling network in Hhip (+/-) mice after CS exposure. CONCLUSIONS: In summary, HHIP haploinsufficiency exaggerated CS-induced airspace enlargement, which models CS-induced emphysema in human smokers carrying COPD risk alleles at the HHIP locus. Network modeling suggested rewired lymphocyte activation signaling circuits in the HHIP haploinsufficiency state.

Progesterone and estradiol synergistically promote the lung metastasis of tuberin-deficient cells in a preclinical model of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-predominant lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis 2 (TSC2) mutations, leading to mTORC1 hyperactivation. The gender specificity of LAM suggests that female hormones contribute to disease progression. Clinical findings indicate that estradiol exacerbates LAM behaviors and symptoms. Although hormonal therapy with progesterone has been employed, the benefit in LAM improvement has not been achieved. We have previously found that estradiol promotes the survival and lung metastasis of cells lacking tuberin in a preclinical model of LAM. In this study, we hypothesize that progesterone alone or in combination with estradiol promotes metastatic behaviors of TSC2-deficient cells. In cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we found that progesterone treatment or progesterone plus estradiol resulted in increased phosphorylation of Protein Kinase B (Akt) and Extracellular signal-regulated kinases1/2 (ERK1/2), induced the proliferation, and enhanced the migration and invasiveness. In addition, treatment of progesterone plus estradiol synergistically decreased the levels of reactive oxygen species and enhanced cell survival under oxidative stress. In a murine model of LAM, treatment of progesterone plus estradiol promoted the growth of xenograft tumors; however, progesterone treatment did not affect the development of xenograft tumors of Tsc2-deficient cells. Importantly, treatment of progesterone plus estradiol resulted in alteration of lung morphology and significantly increased the number of lung micrometastases of Tsc2-deficient cells compared with estradiol treatment alone. Collectively, these data indicate that progesterone increases the metastatic potential of Tsc2-deficient LAM patient-derived cells in vitro and lung metastasis in vivo. Thus, targeting progesterone-mediated signaling events may have therapeutic benefit for LAM and possibly other hormonally dependent cancers.

Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells.

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.

Two mutations impair the stability and function of ubiquitin-activating enzyme (E1).

Protein ubiquitination plays critical roles in the regulation of multiple cellular processes including cell proliferation, signal transduction, oncogenesis, and hypoxic response. TS20 is a Balb3T3-derived cell line in which ubiquitination is inhibited by restrictive temperature. While TS20 has been used to elucidate the degradation of many important proteins including p53, p27, HIF-1α, and ornithine decarboxylase, the molecular basis of its temperature sensitivity has not been fully determined. We cloned full-length E1 cDNA from TS20. Sequencing analysis revealed two point mutations (nt736G to A and nt2313G to C) that lead to substitution of aa189A to T and aa714W to C, respectively. Transient transfection assays revealed that mutant E1 was less stable than its wild-type counterpart, and restrictive temperature (39°C) accelerated its degradation. Under permissive temperature, reverting aa714C to W significantly improved E1 stability and activity. Under restrictive temperature, reverting of both substitutions was required to fully restore E1 stability. Similar results were observed when the mutants were expressed in non-TS20 cells, indicating the mutations are sufficient for its temperature sensitive degradation observed in TS20 cells. Functionally, reverting aa714C to W was sufficient to facilitate the monoubiquitination of H2A and to support TS20 growth at 39°C. It also significantly improved the ubiquitination-dependent disposal of HIF-1α. Our data conclusively demonstrate that mutations introgenic to UVBE1 cause E1 instability, which leads to deficiency of E1 function. Our data establish the molecular basis for unambiguous interpretation of experimental data based on TS20 cells, and provide new insight into the structural determinants of E1 stability.

Nitrogen anabolism underlies the importance of glutaminolysis in proliferating cells.

Glutaminolysis and Warburg effect are the two most noticeable metabolic features of tumor cells whereas their biological significance in cell proliferation remains elusive. A widely accepted current hypothesis is that tumor cells use glutamine as a preferred carbon source for energy and reducing power, which has been used to explain both glutaminolysis and the Warburg effect. Here we provide evidence to show that supplying nitrogen, not the carbon skeleton, underlies the major biological importance of glutaminolysis for proliferating cells. We show alternative nitrogen supplying mechanisms rescue cell proliferation in glutamine-free media. Particularly, we show that ammonia is sufficient to maintain a long-term survival and proliferation of Hep3B in glutamine-free media. We also observed that nitrogen source restriction repressed carbon metabolic pathways including glucose utilization. Based on these new observations and metabolic pathways well established in published literature, we propose an alternative model that cellular demand for glutamate as a key molecule in nitrogen anabolism is the driving force of glutaminolysis in proliferating cells. Our model suggests that the Warburg effect may be a metabolic consequence secondary to the nitrogen anabolism.