Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots

Gabay-Maskit, Shiran; Schuldiner, Maya; Zalckvar, Einat

doi:10.12688/f1000research.13396.1

Cited by 4 publications

(5 citation statements)

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“…To ensure that the peroxisomal localization of Mdh2 and its dependence on Mdh3 and Pex5 is not a result of the GFP tagging, we generated a specific antibody that recognizes Mdh2 but not the other Mdh isoforms (Gabay-Maskit et al, 2018). Analysis of subcellular fractions clearly demonstrated that native and un-tagged Mdh2 also co-sediments with peroxisomes (Figure 3E,control) and that its peroxisomal localization is dependent on the presence of Mdh3 ( Figure 3E, mdh3).…”

Section: The Peroxisomal Localization Of Mdh2 Is Pex5-and Mdh3-dependentmentioning

confidence: 99%

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A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast

Gabay-Maskit

Cruz-Zaragoza

Shai

et al. 2020

Journal of Cell Science

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Eukaryotic cells evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge on molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore critical for understanding eukaryotic metabolism. Here we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. The dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

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Section: The Peroxisomal Localization Of Mdh2 Is Pex5-and Mdh3-dependentmentioning

confidence: 99%

“…The Mdh2 antibody was produced by GeneScript, USA, using a specific Mdh2 peptide as an immunogen: MPHSVTPSIEQDSLC, and was verified by us for specificity and activity (dil. 1:2.500) (Gabay-Maskit et al, 2018…”

Section: Antibodiesmentioning

confidence: 99%

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast

Gabay-Maskit

Cruz-Zaragoza

Shai

et al. 2020

Journal of Cell Science

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“…60 And MDH2 can be identified by specific antibodies with higher and higher specificity. 61 As research showed, cytosolic and mitochondrial MDH activities and cytosolic ratio of MDH/LDH activity in leukocytes from the whole blood of race horses were significantly higher than those of riding horses. It was considered to reflect the elevation of energy metabolism in animal tissues.…”

Section: Is It Feasible To Detect Mdh In Clinic?mentioning

confidence: 93%

Potential biomarkers in septic shock besides lactate

Yang

Zhang

2020

Exp Biol Med (Maywood)

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Septic shock can be defined as sepsis with persisting hypotension and is required for vasopressors after initial unsuccessful fluid resuscitation. Elevated lactate is a biomarker of tissue perfusion and oxygenation and a useful prognostic tool for resuscitation in septic shock, as it is a byproduct of anaerobic glycolysis due to inadequate oxygen delivery and tissue hypoxia. Early and serial systematic lactate measurement will prompt physician more rapid intervention and lactate normalization, which is associated with better outcome. However, lactate formation during septic shock is neither entirely related to tissue hypoxia, nor reversible by increasing oxygen delivery. Meanwhile, lactate can be oxidized via tricarboxylic acid cycle after being transferred into mitochondria via lactate shuttle, which indicates elevated lactate can be used rather than only accumulation. Glycolysis and elevated lactate can be initiated by hypoxia, but persistent hyperlactatemia may not only represent persistent hypoxia. Some other potential biomarkers have been reviewed in the article including intermediates of tricarboxylic acid cycle, malate-aspartate shuttle, the nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD+/NADH) ratio, NAD+, NADH, malate, and malate dehydrogenase from the point of view of energy metabolism. Among them, malate dehydrogenase participates in both malate-aspartate shuttle and tricarboxylic acid cycle, and it can also indirectly reflex the NAD+/NADH ratio. It is reasonable to hypothesize that the combination of lactate and malate dehydrogenase will be more comprehensive to reflex hypoxia in septic shock. Impact statement Elevated lactate has been commonly considered as a biomarker and a useful prognostic tool for resuscitation in septic shock, facilitating physician more rapid intervention and treatment. However, it can be initiated by hypoxia, but persistent hyperlactatemia may not represent persistent hypoxia only. In the article, it is the first time to review potential biomarkers in septic shock from the point of view of energy metabolism including intermediates of TCA cycle, MAS, the NAD+/NADH ratio, NAD+, NADH, malate, and MDH. And the combination of lactate and MDH is also proposed in septic shock for the first time, as MDH in cytoplasm and mitochondria participates in both MAS and TCA cycle for ATP generation. Its feasibility in clinic has been analyzed at the end, although related research is still limited. It is reasonable the combination of lactate and MDH will be more comprehensive to reflex hypoxia in septic shock.

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“…3C). Among them, MDH2 plays a role in cell metabolism and amino acid acetylation [16]. GOT2 is necessary for metabolite exchange between mitochondria and cytoplasm and plays a crucial role in amino acid metabolism [17].…”

Section: Prediction and Analysis Of Interacting Protein With Espfmentioning

confidence: 99%

Identification of a novel interaction between SMC1 DNA damage repair protein and Escherichia coli O157: H7 EspF using co-immunoprecipitation combined with mass spectrometry

Yang

Yan

et al. 2020

Preprint

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Background: It is known that the enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interaction between EspF, its N- or C-terminus, and host proteins, are still unclear. Results: In this study, we used co-immunoprecipitation combined with mass spectrometry to screen EspF-interacting proteins. A total of 311 host proteins are detected. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus of EspF. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. We verify here for the first time that SMC1 interacts with EspF and more strongly with its C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1, known to influence the S-phase cell cycle arrest and usually express during periods of DNA damage. Surprisingly, Mass spectrometry reveals that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage through SMC1 phosphorylation.Conclusion: Taken together, this is the first study describing the interaction between EspF and SMC1. Our work lays a foundation for further research on directly EspF-mediated host cells’ DNA damage, apoptosis, and even colorectal carcinogenesis.

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Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots

Cited by 4 publications

References 10 publications

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast

Potential biomarkers in septic shock besides lactate

Identification of a novel interaction between SMC1 DNA damage repair protein and Escherichia coli O157: H7 EspF using co-immunoprecipitation combined with mass spectrometry

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