comparative-proteomic-analysis-whole-kidney-medulla-cortical-tubules

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a ...

University of Louisville School of Medicine

NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

Patients with systemic lupus erythematosus (SLE) frequently develop lupus nephritis (LN), a complication frequently leading to end stage kidney disease. Immune complex deposition in the glomerulus is central to the development of LN. Using a targeted proteomic approach, we tested the hypothesis that autoantibodies targeting glomerular antigens contribute to the development of LN.

Autoantibodies targeting glomerular annexin A2 identify patients with proliferative lupus nephritis.

A subset of B-cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N-terminal trimethylase NRMT1 and the N-terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide-binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N-terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N-terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ-irradiation.

Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation.

Polychlorinated biphenyl-mediated steatohepatitis has been shown to be due in part to inhibition of epidermal growth factor receptor (EGFR) signalling. EGFR signalling regulates many facets of hepatocyte function, but it is unclear which other kinases and pathways are involved in the development of toxicant-associated steatohepatitis (TASH).

Hepatic signalling disruption by pollutant Polychlorinated biphenyls in steatohepatitis.

Environmental pollution contributes to fatty liver disease pathogenesis. Polychlorinated biphenyl (PCB) exposures have been associated with liver enzyme elevation and suspected steatohepatitis in cohort studies. Male mice treated with the commercial PCB mixture, Aroclor 1260 (20 mg/kg), and fed high fat diet (HFD) for 12 weeks developed steatohepatitis. Receptor-based modes of action including inhibition of the epidermal growth factor (EGF) receptor were previously proposed, but other mechanisms likely exist. Objectives were to identify and validate the pathways, transcription factors, and mechanisms responsible for the steatohepatitis associated with PCB and HFD coexposures. Comparative proteomics analysis was performed in archived mouse liver samples from the aforementioned chronic exposure study. Pathway and transcription factor analysis (TFA) was performed, and selected results were validated. Liver proteomics detected 1103 unique proteins. Aroclor 1260 upregulated 154 and downregulated 93 of these. Aroclor 1260 + HFD coexposures affected 55 pathways including glutathione metabolism, intermediary metabolism, and cytoskeletal remodeling. TFA of Aroclor 1260 treatment demonstrated alterations in the function of 42 transcription factors including downregulation of NRF2 and key nuclear receptors previously demonstrated to protect against steatohepatitis (e.g., HNF4α, FXR, PPARα/δ/γ, etc.). Validation studies demonstrated that Aroclor 1260 significantly reduced HNF4α protein levels, while Aroclor 1260 + HFD reduced expression of the HNF4α target gene, albumin, in vivo. Aroclor 1260 attenuated EGF-dependent HNF4α phosphorylation and target gene activation in vitro. Aroclor 1260 reduced levels of NRF2, its target genes, and glutathione in vivo. Aroclor 1260 attenuated EGF-dependent NRF2 upregulation, in vitro. Aroclor 1260 indirectly activated hepatic stellate cells in vitro via induction of hepatocyte-derived TGFβ. PCB exposures adversely impacted transcription factors regulating liver protection, function, and fibrosis. PCBs, thus, compromised the liver by reducing its protective responses against nutritional stress to promote diet-induced steatohepatitis. The identified mechanisms by which environmental pollutants influence fatty liver disease pathogenesis require confirmation in humans.

Proteomic Analysis Reveals Novel Mechanisms by Which Polychlorinated Biphenyls Compromise the Liver Promoting Diet-Induced Steatohepatitis.

Exposure to arsenic, a class I carcinogen, affects 200 million people globally. Skin is the major target organ, but the molecular etiology of arsenic-induced skin carcinogenesis remains unclear. Arsenite (As)-induced disruption of alternative splicing could be involved, but the mechanism is unknown. Zinc finger proteins play key roles in alternative splicing. As can displace zinc (Zn) from C3H1 and C4 zinc finger motifs (zfm's), affecting protein function. ZRANB2, an alternative splicing regulator with two C4 zfm's integral to its structure and splicing function, was chosen as a candidate for this study. We hypothesized that As could displace Zn from ZRANB2, altering its structure, expression, and splicing function. As/Zn binding and mutual displacement experiments were performed with synthetic apo-peptides corresponding to each ZRANB2 zfm, employing a combination of intrinsic fluorescence, ultraviolet spectrophotometry, zinc colorimetric assay, and liquid chromatography-tandem mass spectrometry. ZRANB2 expression in HaCaT cells acutely exposed to As (0 or 5 μM, 0-72 h; or 0-5 μM, 6 h) was examined by RT-qPCR and immunoblotting. ZRANB2-dependent splicing of TRA2B mRNA, a known ZRANB2 target, was monitored by reverse transcription-polymerase chain reaction. As bound to, as well as displaced Zn from, each zfm. Also, Zn displaced As from As-bound zfm's acutely, albeit transiently. As exposure induced ZRANB2 protein expression between 3 and 24 h and at all exposures tested but not ZRANB2 mRNA expression. ZRANB2-directed TRA2B splicing was impaired between 3 and 24 h post-exposure. Furthermore, ZRANB2 splicing function was also compromised at all As exposures, starting at 100 nm. We conclude that As exposure displaces Zn from ZRANB2 zfm's, changing its structure and compromising splicing of its targets, and increases ZRANB2 protein expression as a homeostatic response both at environmental/toxicological exposures and therapeutically relevant doses.

Arsenite Exposure Displaces Zinc from ZRANB2 Leading to Altered Splicing.

Kidney involvement in systemic lupus erythematosus (SLE)-termed lupus nephritis (LN)-is a severe manifestation of SLE that can lead to end-stage kidney disease (ESKD). LN is characterized by immune complex deposition and inflammation in the glomerulus. We tested the hypothesis that autoantibodies targeting podocyte and glomerular cell proteins contribute to the development of immune complex formation in LN. We used Western blotting with SLE sera from patients with and without LN to identify target antigens in human glomerular and cultured human-derived podocyte membrane proteins. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified the proteins in the gel regions corresponding to reactive bands observed with sera from LN patients. We identified 102 proteins that were present in both the podocyte and glomerular samples. We identified 10 high-probability candidates, including moesin, using bioinformatic analysis. Confirmation of moesin as a target antigen was conducted using immunohistochemical analysis (IHC) of kidney biopsy tissue and enzyme-linked immunosorbent assay (ELISA) to detect circulating antibodies. By IHC, biopsies from patients with proliferative lupus nephritis (PLN, class III/IV) demonstrated significantly increased glomerular expression of moesin ( < 0.01). By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against moesin ( < 0.01). This suggests that moesin is a target glomerular antigen in lupus nephritis.

Patients with Proliferative Lupus Nephritis Have Autoantibodies That React to Moesin and Demonstrate Increased Glomerular Moesin Expression.

Identification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%-20% of patients.

Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy.

Alcohol-associated liver disease (ALD) is a major cause of mortality. Gut barrier dysfunction-induced bacterial translocation and endotoxin release contribute to the pathogenesis of ALD. Probiotic  GG (LGG) is known to be beneficial on experimental ALD by reinforcing the intestinal barrier function. In this study, we aim to investigate whether the protective effects of LGG on intestinal barrier function is mediated by exosome-like nanoparticles (ELNPs) released by LGG. Intestinal epithelial cells and macrophages were treated with LGG-derived ELNPs (LDNPs) isolated from LGG culture. LDNPs increased tight junction protein expression in epithelial cells and protected from the lipopolysaccharide-induced inflammatory response in macrophages. Three-day oral application of LDNPs protected the intestine from alcohol-induced barrier dysfunction and the liver from steatosis and injury in an animal model of ALD. Co-administration of an aryl hydrocarbon receptor (AhR) inhibitor abolished the protective effects of LDNPs, indicating that the effects are mediated, at least in part, by intestinal AhR signaling. We further demonstrated that LDNP administration increased intestinal interleukin-22-Reg3 and nuclear factor erythroid 2-related factor 2 (Nrf2)-tight junction signaling pathways, leading to the inhibition of bacterial translocation and endotoxin release in ALD mice. This protective effect was associated with LDNP enrichment of bacterial tryptophan metabolites that are AhR agonists.  Our results suggest that the beneficial effects of LGG and their supernatant in ALD are likely mediated by bacterial AhR ligand-enriched LDNPs that increase Reg3 and Nrf2 expression, leading to the improved barrier function. These findings provide a strategy for the treatment of ALD and other gut barrier dysfunction-associated diseases.

Exosome-Like Nanoparticles From GG Protect Against Alcohol-Associated Liver Disease Through Intestinal Aryl Hydrocarbon Receptor in Mice.

An elevated expression of phosphoserine aminotransferase 1 (PSAT1) has been observed in multiple tumor types and is associated with poorer clinical outcomes. Although PSAT1 is postulated to promote tumor growth through its enzymatic function within the serine synthesis pathway (SSP), its role in cancer progression has not been fully characterized. Here, we explore a putative non-canonical function of PSAT1 that contributes to lung tumor progression. Biochemical studies found that PSAT1 selectively interacts with pyruvate kinase M2 (PKM2). Amino acid mutations within a PKM2-unique region significantly reduced this interaction. While PSAT1 loss had no effect on cellular pyruvate kinase activity and PKM2 expression in non-small-cell lung cancer (NSCLC) cells, fractionation studies demonstrated that the silencing of PSAT1 in epidermal growth factor receptor (EGFR)-mutant PC9 or EGF-stimulated A549 cells decreased PKM2 nuclear translocation. Further, PSAT1 suppression abrogated cell migration in these two cell types whereas PSAT1 restoration or overexpression induced cell migration along with an elevated nuclear PKM2 expression. Lastly, the nuclear re-expression of the acetyl-mimetic mutant of PKM2 (K433Q), but not the wild-type, partially restored cell migration in PSAT1-silenced cells. Therefore, we conclude that, in response to EGFR activation, PSAT1 contributes to lung cancer cell migration, in part, by promoting nuclear PKM2 translocation.

Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells.

Chronic kidney disease (CKD) affects 30 million adults, costs ~$79 billion dollars (2016) in Medicare expenditures, and is the ninth leading cause of death in the United States. The disease is silent or undiagnosed in almost half of people with severely reduced kidney function. Urine provides an ideal biofluid that is accessible to high-sensitivity mass spectrometry-based proteomic interrogation and is an indicator of renal homeostasis. While the accurate and precise diagnosis and better disease management of CKD can be aided using urine biomarkers, their discovery in excessive protein or nephrotic urine samples can present challenges. In this work we present a mass spectrometry-based method utilizing multiplex tandem mass tag (TMT) quantification and improved protein quantification using reporter ion normalization to urinary creatinine to analyze urinary proteins from patients with a form of nephrotic syndrome (FSGS). A comparative analysis was performed for urine from patients in remission versus active disease flare. Two-dimensional LC-MS/MS TMT quantitative analysis identified over 1058 urine proteins, 580 proteins with 2 peptides or greater and quantifiable. Normalization of TMT abundance values to creatinine per ml of urine concentrated reduced variability in 2D-TMT-LC-MS/MS experiments. Univariate and multivariate analyses showed that 27 proteins were significantly increased in proteinuric disease flare. Hierarchical heatmap clustering showed that SERPINA1 and ORM1 were >1.5 fold increased in active disease versus remission urine samples. ELISA validation of SERPINA1 and ORM1 abundance agreed with our quantitative TMT proteomics analysis. These findings provide support for the utility of this method for identification of novel diagnostic markers of CKD and identify SERPINA1 and ORM1 as promising candidate diagnostic markers for FSGS.

Quantitative Mass Spectrometry Normalization in Urine Biomarker Analysis in Nephrotic Syndrome.

Arylamine -acetyltransferase 1 () is frequently upregulated in breast cancer. An unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate  knockout (KO) cell lines were performed. Among 4,890 proteins identified, 737 and 651 proteins were found significantly ( < 0.01) upregulated and downregulated, respectively, in  KO cells, compared to the parental cells. Each set of proteins was analyzed to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in the set. Among the proteins upregulated in  KO cells, processes associated with MHC major histocompatibility complex I-mediated antigen presentation were significantly enriched. Multiple processes involved in mitochondrial functions were collectively downregulated in  KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in  KO cells. The current dataset contains additional representations of the biological processes and components that are differentially enriched in  KO MDA-MB-231 cells and will serve as a basis for generating novel hypotheses regarding the role of NAT1 in breast cancer. Data are available via ProteomeXchange with identifier PXD035953.

Dataset for proteomic analysis of arylamine -acetyltransferase 1 knockout MDA-MB-231 breast cancer cells.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) continues to increase due in part to the obesity epidemic and to environmental exposures to metabolism disrupting chemicals. A single gavage exposure of male mice to Aroclor 1260 (Ar1260), an environmentally relevant mixture of non-dioxin-like polychlorinated biphenyls (PCBs), resulted in steatohepatitis and altered RNA modifications in selenocysteine tRNA 34 weeks post-exposure. Unbiased approaches identified the liver proteome, selenoproteins, and levels of 25 metals. Ar1260 altered the abundance of 128 proteins. Enrichment analysis of the liver Ar1260 proteome included glutathione metabolism and translation of selenoproteins. Hepatic glutathione peroxidase 4 (GPX4) and Selenoprotein O (SELENOO) were increased and Selenoprotein F (SELENOF), Selenoprotein S (SELENOS), Selenium binding protein 2 (SELENBP2) were decreased with Ar1260 exposure. Increased copper, selenium (Se), and zinc and reduced iron levels were detected. These data demonstrate that Ar1260 exposure alters the (seleno)proteome, Se, and metals in MASLD-associated pathways.

Article	Year
NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1. The Biochemical journal\| PubMed ID: 24090352	2013
Autoantibodies targeting glomerular annexin A2 identify patients with proliferative lupus nephritis. Proteomics. Clinical applications\| PubMed ID: 25824007	2015
Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation. Protein science : a publication of the Protein Society\| PubMed ID: 28556566	2017
Hepatic signalling disruption by pollutant Polychlorinated biphenyls in steatohepatitis. Cellular signalling\| PubMed ID: 30300668	2019
Proteomic Analysis Reveals Novel Mechanisms by Which Polychlorinated Biphenyls Compromise the Liver Promoting Diet-Induced Steatohepatitis. Journal of proteome research\| PubMed ID: 30807179	2019
Arsenite Exposure Displaces Zinc from ZRANB2 Leading to Altered Splicing. Chemical research in toxicology\| PubMed ID: 32274925	2020
Patients with Proliferative Lupus Nephritis Have Autoantibodies That React to Moesin and Demonstrate Increased Glomerular Moesin Expression. Journal of clinical medicine\| PubMed ID: 33669337	2021
Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy. Journal of the American Society of Nephrology : JASN\| PubMed ID: 33952630	2021
Exosome-Like Nanoparticles From GG Protect Against Alcohol-Associated Liver Disease Through Intestinal Aryl Hydrocarbon Receptor in Mice. Hepatology communications\| PubMed ID: 34027273	2021
Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells. Cancers\| PubMed ID: 34439090	2021
Quantitative Mass Spectrometry Normalization in Urine Biomarker Analysis in Nephrotic Syndrome. Glomerular diseases\| PubMed ID: 36199623	2022
Dataset for proteomic analysis of arylamine -acetyltransferase 1 knockout MDA-MB-231 breast cancer cells. Data in brief\| PubMed ID: 36426076	2022
Chronic Aroclor 1260 exposure alters the mouse liver proteome, selenoproteins, and metals in steatotic liver disease. Environmental toxicology and pharmacology\| PubMed ID: 38552755	2024

Daniel W. Wilkey

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