Zymography methods for visualizing hydrolytic enzymes
TL;DR: Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.
Abstract: Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.
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TL;DR: In Mmp9 gene knockout mice, specific spontaneous phenotypes emerged with effects on the skeletal, reproductive and nervous systems and stimulate research on the roles of MMPs and MMP-9 in endocrinology, immunology and the neurosciences.
Abstract: Research on matrix metalloproteinases (MMPs) and in particular on gelatinase B, alias MMP-9, has grown exponentially in the decade 2003–2012. Structural details about flexibility of MMP-9 monomers, together with glycosylation, oligomerization, heterogeneity and instability of the wildtype enzyme explain why crystallography experiments have not yet been successful for the intact enzyme. MMP-9 may be viewed as a multidomain enzyme in which the hemopexin, the O-glycosylated and the catalytic domains yield support for attachment, articulation and catalysis, respectively. The stepwise proteolytic activation of the inactive zymogen into a catalytically active form becomes gradually better understood. Priming of activation by MMP-3 may be executed by meprins that destabilize the interaction of the aminoterminus with the third fibronectin repeat. Alternatively, autocatalytic activation may occur in the presence of molecules that tightly bind to the catalytic site and that push the cystein residue in the p...
604 citations
Cites background or methods from "Zymography methods for visualizing ..."
...A negative point, about publications in which zymography is used, is the classical misinterpretation that enzyme activities were measured (Vandooren et al., 2013)....
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...…intuition and as misinterpreted in many studies on biological samples, gelatin substrate zymography analysis does not yield information on enzyme activity, because it also detects (inactive) proforms and because MMP-9/TIMP-1 complexes dissociate during electrophoresis (Vandooren et al., 2013)....
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...Although this picture is skewed and is a result, probably an artifact, of the commonly used and picogramsensitive technology of gelatin zymography (Vandooren et al., 2013), it also implies that filtering the literature to generate holistic insights or generally applicable paradigms in the MMP field…...
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...If the SDS can be removed completely during the renaturation process and pro-MMP-9 refolds completely, then the zymogen form is not visible anymore on zymography (Vandooren et al., 2013)....
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...Indeed zymography analysis, eventually after sample prepurification (Descamps et al., 2002), gives information about all molecular forms, including monomers, multimers, activation and degradation products, but is in most cases a semi-quantitative method (Vandooren et al., 2013)....
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TL;DR: A previously unexplored role for early BBB disruption in stroke outcomes is identified, whereby BBB rupture may be a cause rather than a consequence of parenchymal cell injury.
Abstract: The mechanism and long-term consequences of early blood-brain barrier (BBB) disruption after cerebral ischaemic/reperfusion (I/R) injury are poorly understood. Here we discover that I/R induces subtle BBB leakage within 30-60 min, likely independent of gelatinase B/MMP-9 activities. The early BBB disruption is caused by the activation of ROCK/MLC signalling, persistent actin polymerization and the disassembly of junctional proteins within microvascular endothelial cells (ECs). Furthermore, the EC alterations facilitate subsequent infiltration of peripheral immune cells, including MMP-9-producing neutrophils/macrophages, resulting in late-onset, irreversible BBB damage. Inactivation of actin depolymerizing factor (ADF) causes sustained actin polymerization in ECs, whereas EC-targeted overexpression of constitutively active mutant ADF reduces actin polymerization and junctional protein disassembly, attenuates both early- and late-onset BBB impairment, and improves long-term histological and neurological outcomes. Thus, we identify a previously unexplored role for early BBB disruption in stroke outcomes, whereby BBB rupture may be a cause rather than a consequence of parenchymal cell injury.
297 citations
TL;DR: Findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity.
189 citations
Cites methods from "Zymography methods for visualizing ..."
...Analysis of MMP-9 Activity by Zymography Zymograms were performed as described previously (Vandooren et al., 2013)....
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...We tested this by measuring secretion of MMP-9 by neutrophils in Tnfr / and wild-type mice by zymography (Vandooren et al., 2013)....
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...Zymograms were performed as described previously (Vandooren et al., 2013)....
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TL;DR: This Review discusses the structural biology aspects and mechanisms of catalysis by different protease classes, and provides an overview of biological pathways that utilize specific proteolytic cleavage as a precision control mechanism in protein quality control, stability, localization, and maturation, as well as proteolytes as a mediator in signaling pathways.
Abstract: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissue—from 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C-termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the s...
139 citations
TL;DR: The shape and extent of the rhizosphere for enzyme activities is plant species specific and varies due to differentrhizosphere processes and functions (e.g. root exudation) and functions and should be considered in assessments and modeling of rhizospheric extension.
Abstract: The rhizosphere, the small soil volume that surrounds and is influenced by plant roots, is one of the most dynamic biological interfaces on Earth. Enzymes, produced by both roots and microorganisms, are the main biological drivers of SOM decomposition. In situ soil zymography was applied to test hypotheses that 1) the spatial pattern of rhizosphere activity is enzyme-specific and 2) the distribution of enzyme activity along the roots is dependent on root system and plant species. Lentil (Lens culinaris) and maize (Zea mays L.), two species with contrasting root physiology, were chosen to test their effects on spatial distribution of activities of β-glucosidase, cellobiohydrolase, leucine-aminopeptidase and phosphatase. The extent of the rhizosphere for each enzyme and plant species was estimated as a function of distance from the root. For the first time, we demonstrated plant-specific patterns of exoenzyme distribution: these were uniform along the lentil roots, whereas in the rhizosphere of maize, the enzyme activities were higher at the apical or proximal root parts. We conclude that the shape and extent of the rhizosphere for enzyme activities is plant species specific and varies due to different rhizosphere processes (e.g. root exudation) and functions (e.g. nutrient mobilization abilities). The extension of enzyme activity into the rhizosphere soil was minimal (1 mm) for enzymes responsible for the C cycle and maximal (3.5 mm) for enzymes of the phosphorus cycle. This should be considered in assessments and modeling of rhizosphere extension and the corresponding effects on soil properties and functions.
128 citations
Additional excerpts
...Zymography, a non-destructive in situ technique for twodimensional imaging, now offers an opportunity for visualization of enzyme activities -spatial and temporal- in soil and in the rhizosphere (Spohn and Kuzyakov, 2013, 2014; Vandooren et al., 2013)....
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References
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TL;DR: A method is devised which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections and may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.
Abstract: Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvasculature of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.
2,503 citations
TL;DR: A new technique is described for the electrophoretic analysis of plasminogen activators in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized pl asminogen and gelatin, which can be used to detect as little as 1 mU of urokinase and effectively distinguishes between melanoma- and u rokinase-type plasmineg activators.
1,988 citations
TL;DR: In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters, and this strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.
Abstract: We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.
1,695 citations
TL;DR: The model suggests that lipid plays an essential role and show that even substances which are not intrinsically surface active can have profound effects when the composition of the interfacial phase is suitably chosen, and further studies of its changes of physical state under narcotics may be very informative.
Abstract: ing from this point of view, one could say that we have built and tested a crude membrane model in which such a phenomenon might be observed, and that thus far the binding energies agree satisfactorily with those calculated from in vivo experiments only when the model contains lipid. In so far as we understand the behavior of the model, it does not permit us to state whether the interactions occur primarily between lipid film and narcotic or between water and narcotic, facilitated by the presence of lipid. In either interpretation, our data suggest that lipid plays an essential role and show that even substances which are not intrinsically surface active can have profound effects when the composition of the interfacial phase is suitably chosen.\"7 The sensitivity of the model to inert gases and the quantitative similarity of its reactions to their biological effect suggest that further studies of its changes of physical state under narcotics may be very informative.
1,234 citations
1,152 citations