Development of a Fluorescence Resonance Energy Transfer Peptide Library Technology for Detection of Protease Contaminants in Protein-Based Raw Materials Used in Diagnostic Assays

Kapprell, Hans-Peter; Maurer, Andreas; Kramer, Florian; Heinrich, Boris; Buenning, Carsten; Narváez, Alfredo R.; Kalbacher, Hubert; Flad, Thomas

doi:10.1089/adt.2010.0360

Cited by 9 publications

(6 citation statements)

References 11 publications

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“…Proteasuria was also shown to occur in urine samples from patients with nephrotic syndrome of a different etiology. Proteasuria was quantified using a universal protease substrate library and serine proteases were identified using proteomic profiling . In urine samples from nephrotic patients, total proteolytic activity was increased compared to that of healthy control persons .…”

Section: Proteasuria In Patients With Nephrotic Syndromementioning

confidence: 99%

Proteasuria—The impact of active urinary proteases on sodium retention in nephrotic syndrome

Artunc¹,

Wörn²,

Schork³

et al. 2019

Acta Physiologica

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Sodium retention and extracellular volume expansion are typical features of patients with nephrotic syndrome. In recent years, from in vitro data, endoluminal activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases has been proposed as an underlying mechanism. Recently, this concept was supported in vivo in nephrotic mice that were protected from proteolytic ENaC activation and sodium retention by the use of aprotinin for the pharmacological inhibition of urinary serine protease activity. These and other findings from studies in both rodents and humans highlight the impact of active proteases in the urine, or proteasuria, on ENaC-mediated sodium retention and edema formation in nephrotic syndrome. Targeting proteasuria could become a therapeutic approach to treat patients with nephrotic syndrome. However, pathophysiologically relevant proteases remain to be identified. In this review, we introduce the concept of proteasuria to explain tubular sodium avidity and conclude that proteasuria can be considered as a key mechanism of sodium retention in patients with nephrotic syndrome. K E Y W O R D Saprotinin, ENaC, nephrotic syndrome, proteasuria, proteinuria, proteolysis, serine protease

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Section: Proteasuria In Patients With Nephrotic Syndromementioning

confidence: 99%

Proteasuria—The impact of active urinary proteases on sodium retention in nephrotic syndrome

Artunc¹,

Wörn²,

Schork³

et al. 2019

Acta Physiologica

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“…Modern proteomics has matured to provide deep maps of protein compositions (Rinschen et al 2018a ) and is able to be paired with high-throughput automation (Müller et al 2020 ) and quality control systems important for clinical application (Dayon et al 2014 ). Alternatively, routine clinical assaying to quantify specific proteases as protein markers, upregulated in response to disease, can be performed with immunometric assays (Kapprell et al 2011 ). While the qualification and quantification of proteases are indispensable, full characterization of protease networks also requires functional assessment of protease activity.…”

Section: Novel Analytical Approaches To Map Proteolysis In Vivomentioning

confidence: 99%

“…The fluorescent labels only fluoresce if the pentapeptide is proteolyzed (Enari et al 1996 ). Following cleavage of the pentapeptide by a protease, the quencher DNP is removed, and MCA emits fluorescence, which can be quantified (Enari et al 1996 ; Kapprell et al 2011 ). Addition of different protease inhibitors enables to pinpoint the protease activity to specific protease classes.…”

Section: Novel Analytical Approaches To Map Proteolysis In Vivomentioning

confidence: 99%

“…Additional proteases found to be active and at elevated concentration in the urine are plasmin (Svenningsen et al 2009 ) and prostasin/kallikrein (Zachar et al 2015 ). The Artunc group has catalogued the proteases active in urine from patients with acute nephrotic syndrome and a corresponding mouse model (Wörn et al 2021 ) by nLC-MS/MS and in a fluorescence-based assay (Kapprell et al 2011 ) (Fig. 3 c).…”

Section: Global Proteolytic Analysis In Glomerular Diseasementioning

confidence: 99%

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Proteolysis and inflammation of the kidney glomerulus

et al. 2021

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Proteases play a central role in regulating renal pathophysiology and are increasingly evaluated as actionable drug targets. Here, we review the role of proteolytic systems in inflammatory kidney disease. Inflammatory kidney diseases are associated with broad dysregulations of extracellular and intracellular proteolysis. As an example of a proteolytic system, the complement system plays a significant role in glomerular inflammatory kidney disease and is currently under clinical investigation. Based on two glomerular kidney diseases, lupus nephritis, and membranous nephropathy, we portrait two proteolytic pathomechanisms and the role of the complement system. We discuss how profiling proteolytic activity in patient samples could be used to stratify patients for more targeted interventions in inflammatory kidney diseases. We also describe novel comprehensive, quantitative tools to investigate the entirety of proteolytic processes in a tissue sample. Emphasis is placed on mass spectrometric approaches that enable the comprehensive analysis of the complement system, as well as protease activities and regulation in general.

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“…To clarify whether XopJ acts as a protease itself, protease activity was monitored using a Förster resonance energy transfer-based protease detection kit containing a substrate peptide library of more than 2 3 10 6 peptide variants, with proteinase K serving as a positive control (Kapprell et al, 2011). Recombinant Maltose-Binding Protein (MBP)-XopJ, purified from Escherichia coli, showed a significant increase in protease activity when compared with MBP alone (Fig.…”

Section: Xopj Possesses Protease Activity In Vitro and In Vivomentioning

confidence: 99%

The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT6

Üstün

Börnke

2015

Plant Physiol.

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Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities.

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Development of a Fluorescence Resonance Energy Transfer Peptide Library Technology for Detection of Protease Contaminants in Protein-Based Raw Materials Used in Diagnostic Assays

Cited by 9 publications

References 11 publications

Proteasuria—The impact of active urinary proteases on sodium retention in nephrotic syndrome

Proteasuria—The impact of active urinary proteases on sodium retention in nephrotic syndrome

Proteolysis and inflammation of the kidney glomerulus

The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT6

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