An automated assay of urinary alanine aminopeptidase activity

Holdt, Birgit; Peters, Eva M.J.; Nagel, Heinz-Rudolf; Steiner, Michael

doi:10.1515/cclm.2008.103

Cited by 5 publications

(7 citation statements)

References 17 publications

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“…Studies have shown that kidney involvement is frequent in COVID-19 patients and even acute kidney injury is common in critically ill patients (56). Our interactome analysis showed an interaction of ORF7a and nsp8 with NPEPPS, a puromycin-sensitive amino peptidase, which is commonly used as a biomarker against damaged kidneys (57). Our results also indicated the interaction of ORF7a and nsp8 with multiple proteasome-related proteins such as PSMD6, PSMD7, PSMD2, and PSMD13, which is in agreement with a previous study in which the authors revealed the interactions by overexpressing the SARS-CoV-2 gene in HEK293 cells (58).…”

Section: Resultsmentioning

confidence: 62%

Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Reveals Mosaic Pattern of Phylogeographical Distribution

et al. 2020

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The outbreak of coronavirus disease 2019 (COVID-19) that started in Wuhan, China, in December 2019 has spread worldwide, emerging as a global pandemic. The severe respiratory pneumonia caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has so far claimed more than 0.38 million lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 virus displays high transmission rates, the underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries, suggesting that its spread is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nonstructural protein 13 (nsp13), nsp14, nsp15, and nsp16 (ORF1b polyproteins) and in the S-protein within 10 viral isolates from the United States. These viral proteins are involved in RNA replication and binding with the human receptors, indicating that the viral variants that are circulating in the population of the United States are different from those circulating in the populations of other countries. In addition, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of a U.S. isolate (GenBank accession no. MT188341.1) leading to a shift in the amino acid frame from position 2540 onward. Through comparative structural analysis of the wild-type and mutant proteins, we showed that this addition of a phenylalanine residue renders the protein in the mutant less stable, which might affect mRNA cap-1 methyltransferase function. We further analyzed the SARS-CoV-2–human interactome, which revealed that the interferon signaling pathway is targeted by orf1ab during infection and that it also interacts with NF-κB-repressing factor (NKRF), which is a potential regulator of interleukin-8 (IL-8). We propose that targeting this interaction may subsequently improve the health condition of COVID-19 patients. Our analysis also emphasized that SARS-CoV-2 manipulates spliceosome machinery during infection; hence, targeting splicing might affect viral replication. In conclusion, the replicative machinery of SARS-CoV-2 is targeting interferon and the notch signaling pathway along with spliceosome machinery to evade host challenges. IMPORTANCE The COVID-19 pandemic continues to storm the world, with over 6.5 million cases worldwide. The severity of the disease varies with the territories and is mainly influenced by population density and age factor. In this study, we analyzed the transmission pattern of 95 SARS-CoV-2 genomes isolated from 11 different countries. Our study also revealed several nonsynonymous mutations in ORF1b and S-proteins and the impact on their structural stability. Our analysis showed the manipulation of host system by viral proteins through SARS-CoV-2–human protein interactome, which can be useful to understand the impact of virus on human health.

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Section: Resultsmentioning

confidence: 62%

Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Reveals Mosaic Pattern of Phylogeographical Distribution

et al. 2020

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“…They have an obvious diagnostic benefit because their measurements may provide detailed information about the nature, size and site of the damage to tubular cells and their possible necrosis or dysfunction [5]. One of these enzymes, AlaAp (EC 3.4.11.2), is an enzyme of the brush border proposed in the early seventies as a urinary marker of renal disease [6], and there is an automatic photometric assay for its determination [7]. AlaAp, together with GluAp (EC 3.4.11.7), CysAp (3.4.11.3) and AspAp (EC 3.4.11.21) are present in the renal tubular cells [8]–[10] and they have an aminopeptidasic function in angiotensin II metabolism, peptide that is increased in renal diseases.…”

Section: Introductionmentioning

confidence: 99%

Urinary Aminopeptidase Activities as Early and Predictive Biomarkers of Renal Dysfunction in Cisplatin-Treated Rats

et al. 2012

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This study analyzes the fluorimetric determination of alanyl- (Ala), glutamyl- (Glu), leucyl-cystinyl- (Cys) and aspartyl-aminopeptidase (AspAp) urinary enzymatic activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats. Male Wistar rats (n = 8 each group) received a single subcutaneous injection of either saline or cisplatin 3.5 or 7 mg/kg, and urine samples were taken at 0, 1, 2, 3 and 14 days after treatment. In urine samples we determined Ala, Glu, Cys and AspAp activities, proteinuria, N-acetyl-β-D-glucosaminidase (NAG), albumin, and neutrophil gelatinase-associated lipocalin (NGAL). Plasma creatinine, creatinine clearance and renal morphological variables were measured at the end of the experiment. CysAp, NAG and albumin were increased 48 hours after treatment in the cisplatin 3.5 mg/kg treated group. At 24 hours, all urinary aminopeptidase activities and albuminuria were significantly increased in the cisplatin 7 mg/kg treated group. Aminopeptidase urinary activities correlated (p<0.011; r2>0.259) with plasma creatinine, creatinine clearance and/or kidney weight/body weight ratio at the end of the experiment and they could be considered as predictive biomarkers of renal injury severity. ROC-AUC analysis was made to study their sensitivity and specificity to distinguish between treated and untreated rats at day 1. All aminopeptidase activities showed an AUC>0.633. We conclude that Ala, Cys, Glu and AspAp enzymatic activities are early and predictive urinary biomarkers of the renal dysfunction induced by cisplatin. These determinations can be very useful in the prognostic and diagnostic of renal dysfunction in preclinical research and clinical practice.

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“…The measurement of urinary enzymes and other urinary biomarkers may therefore be a valuable tool to obtain an early diagnosis during initial stages of renal disease and to follow its progression or regression, facilitating prediction of the prognosis. Urinary APs are considered promising and useful biomarkers of renal disease of different pathophysiological origin, and an automated photometric assay has been developed for their measurement [ 94 ].…”

Section: Aps As Urinary Biomarkers Of Renal Injurymentioning

confidence: 99%

“…APs activities can be readily quantified in biological samples by kinetic methods using photometric [ 94 ] or fluorometric [ 113 ] assays. Widely applied immunological techniques such as immunoblotting or ELISA can be used to analyze the amount/expression of these proteins in biological samples.…”

Section: Quantification Of Urinary Apsmentioning

confidence: 99%

Aminopeptidases in Cardiovascular and Renal Function. Role as Predictive Renal Injury Biomarkers

Vargas

Wangesteen

Rodríguez-Gómez

et al. 2020

IJMS

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Aminopeptidases (APs) are metalloenzymes that hydrolyze peptides and polypeptides by scission of the N-terminus amino acid and that also participate in the intracellular final digestion of proteins. APs play an important role in protein maturation, signal transduction, and cell-cycle control, among other processes. These enzymes are especially relevant in the control of cardiovascular and renal functions. APs participate in the regulation of the systemic and local renin–angiotensin system and also modulate the activity of neuropeptides, kinins, immunomodulatory peptides, and cytokines, even contributing to cholesterol uptake and angiogenesis. This review focuses on the role of four key APs, aspartyl-, alanyl-, glutamyl-, and leucyl-cystinyl-aminopeptidases, in the control of blood pressure (BP) and renal function and on their association with different cardiovascular and renal diseases. In this context, the effects of AP inhibitors are analyzed as therapeutic tools for BP control and renal diseases. Their role as urinary biomarkers of renal injury is also explored. The enzymatic activities of urinary APs, which act as hydrolyzing peptides on the luminal surface of the renal tubule, have emerged as early predictive renal injury biomarkers in both acute and chronic renal nephropathies, including those induced by nephrotoxic agents, obesity, hypertension, or diabetes. Hence, the analysis of urinary AP appears to be a promising diagnostic and prognostic approach to renal disease in both research and clinical settings.

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An automated assay of urinary alanine aminopeptidase activity

Abstract: We conclude that the newly developed automated assay represents a user-friendly and analytical tool to determine urinary AAP activity in a medium- to high-throughput laboratory. Potential applications include clinical diagnostic work and toxicological studies.

Cited by 5 publications

References 17 publications

Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Reveals Mosaic Pattern of Phylogeographical Distribution

Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Reveals Mosaic Pattern of Phylogeographical Distribution

Urinary Aminopeptidase Activities as Early and Predictive Biomarkers of Renal Dysfunction in Cisplatin-Treated Rats

Aminopeptidases in Cardiovascular and Renal Function. Role as Predictive Renal Injury Biomarkers

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