Valery Yermalitsky - Academia.edu (original) (raw)

Papers by Valery Yermalitsky

Alzheimers & Dementia, Jul 1, 2006

ELISA-type assays, allowed the isolation of a panel of Nanobodies with different binding-capacity... more ELISA-type assays, allowed the isolation of a panel of Nanobodies with different binding-capacity and specificity. Selected Nanobodies were characterized for their biochemical and immuno-histochemical specificity, respectively by Western blotting and on brain sections of APP[V717I] transgenic mice, our validated model for amyloid pathology in AD. The combination of classical and urea/SDS-PAGE with immunoblotting and histo-amyloid-reactivity resulted in the selection of several Nanobodies differing in specificity, i.e. reacting with all A , with A 42, and/or with amyloid plaques. The latter was confirmed in vivo by intra-cerebral injection. Conclusions: Based on the selected Nanobodies, we derived bivalent and bispecific Nanobodies that are directed to different molecular targets, as well as others that can actively pass the blood-brain barrier, aiming at therapeutic and diagnostic purposes.

JCI Insight

Text, References, Tables and Figures Legends): 9296

Data for development of simplified assay for isolevuglandin protein adducts and final protocol.

Arteriosclerosis, Thrombosis, and Vascular Biology, May 1, 2019

Structure (London, England : 1993), 2006

NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of ... more NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between ...

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arach... more The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to gen-erate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosa-trienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, re-constituted in the presence of [1-14C]arachidonic acid. The majority of the 14C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygen-ase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product

This article has not been copyedited and formatted. The final version may differ from this v rsion.

ABSTRACTThe lipid aldehyde 4-oxo-2-nonenal (ONE) derived from peroxidation of n-6 polyunsaturated... more ABSTRACTThe lipid aldehyde 4-oxo-2-nonenal (ONE) derived from peroxidation of n-6 polyunsaturated fatty acids and generated in parallel to 4-hydroxynonenal (HNE) is a highly reactive protein crosslinker. Crosslinking of proteins in high-density lipoprotein (HDL) by lipid peroxidation products causes HDL dysfunction and contributes to atherogenesis. While HNE is relatively well studied, the relevance of ONE in atherosclerosis and in modifying HDL has not been examined. In the present study, we found a significant increase in ONE-ketoamide (lysine) adducts in HDL derived from patients with familial hypercholesterolemia (FH) (1620 ± 985.4 pmol/mg) compared to healthy controls (664 ± 219.5 pmol/mg). ONE crosslinked apoA-I on HDL at a concentration of >3 mol ONE per 10 mol apoA-I (0.3 eq), which is 100-fold lower than HNE but comparable to the potent protein crosslinker, isolevuglandin. ONE-modified HDL partially inhibited the ability of HDL to protect against LPS-induced TNFα and IL-...

The epidermis expresses cyclooxygenases, lipoxygenases and cytochromes P450, which utilize arachi... more The epidermis expresses cyclooxygenases, lipoxygenases and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12-and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-14 C]-arachidonic acid. The majority of the [ 14 C]products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) co-eluted with the CYP2B19 product 14,15-EET on a reversed-phase HPLC system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal [ 14 C]-products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPHdependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic This article has not been copyedited and formatted. The final version may differ from this version.

Alzheimer's & Dementia, 2008

Alzheimer's & Dementia, 2006

Protein …, 2006

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo... more Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification ...

Hypertension, 2020

Supplemental Digital Content is available in the text. Hypertension remains a major health proble... more Supplemental Digital Content is available in the text. Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochond...

The pathogenesis of atherosclerosis is accelerated by oxidative stress, which produces lipid pero... more The pathogenesis of atherosclerosis is accelerated by oxidative stress, which produces lipid peroxidation. Among the products of lipid peroxidation are highly reactive dicarbonyls that covalently modify proteins. We investigated the impact of treatment with the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in hyperlipidemic Ldlr-/- mice, a model of familial hypercholesterolemia (FH). Compared to mice treated with vehicle, 2-HOBA significantly decreased atherosclerosis in hypercholesterolemic Ldlr-/- mice by 31% in the proximal aortas and 60% in en face aortas, in the absence of changes in blood lipid levels. 2-HOBA reduced malondialdehyde (MDA) content in HDL, LDL, and in the atherosclerotic lesions. Consuming a Western diet increased plasma MDA-apoAI adduct levels in Ldlr-/- mice. 2-HOBA inhibited MDA-apoAI formation and increased the capacity of the mouse HDL to reduce macrophage cholesterol stores. Furthermore, 2-HOBA diminished oxidative...

Archives of Biochemistry and Biophysics, 2005

CYP2B19 is an arachidonic acid monooxygenase highly expressed in the outer, diVerentiated cell la... more CYP2B19 is an arachidonic acid monooxygenase highly expressed in the outer, diVerentiated cell layers of mouse epidermis. We aimed to establish whether CYP2B19 is the source of epidermal epoxyeicosatrienoic acids (EETs), which are implicated in mechanisms regulating epidermal corniWcation. We show that primary cultures of mouse epidermal keratinocytes expressed native CYP2B19, as determined by mass spectrometry. DiVerentiation upregulated CYP2B19 mRNA levels (t39-fold) detected by real-time PCR, CYP2B19 immunoreactivity detected by Western blotting, and cellular levels of the CYP2B19 product 11,12-EET. Cellular 11,12-EET formed from endogenous arachidonic acid increased preferentially (4-to 12-fold) at Day 4 or 5 of diVerentiation, compared with undiVerentiated (Day 0) keratinocyte cultures. Temporally, these results concur with the maximal levels of CYP2B19 mRNA measured at Day 2 and CYP2B19 immunoreactivity at Day 4. We conclude that while mouse epidermis likely expresses multiple cytochrome P450 enzymes, existing evidence supports native CYP2B19 as being the major source of epidermal EET formation.

Obesity Research & Clinical Practice

Alzheimer's & Dementia, 2006

Protein Science, 2006

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo... more Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.

Journal of Pharmacology and Experimental Therapeutics, 2005

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arach... more The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved th...

Journal of Biological Chemistry

Alzheimers & Dementia, Jul 1, 2006

ELISA-type assays, allowed the isolation of a panel of Nanobodies with different binding-capacity... more ELISA-type assays, allowed the isolation of a panel of Nanobodies with different binding-capacity and specificity. Selected Nanobodies were characterized for their biochemical and immuno-histochemical specificity, respectively by Western blotting and on brain sections of APP[V717I] transgenic mice, our validated model for amyloid pathology in AD. The combination of classical and urea/SDS-PAGE with immunoblotting and histo-amyloid-reactivity resulted in the selection of several Nanobodies differing in specificity, i.e. reacting with all A , with A 42, and/or with amyloid plaques. The latter was confirmed in vivo by intra-cerebral injection. Conclusions: Based on the selected Nanobodies, we derived bivalent and bispecific Nanobodies that are directed to different molecular targets, as well as others that can actively pass the blood-brain barrier, aiming at therapeutic and diagnostic purposes.

JCI Insight

Text, References, Tables and Figures Legends): 9296

Data for development of simplified assay for isolevuglandin protein adducts and final protocol.

Arteriosclerosis, Thrombosis, and Vascular Biology, May 1, 2019

Structure (London, England : 1993), 2006

NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of ... more NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between ...

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arach... more The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to gen-erate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosa-trienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, re-constituted in the presence of [1-14C]arachidonic acid. The majority of the 14C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygen-ase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product

This article has not been copyedited and formatted. The final version may differ from this v rsion.

ABSTRACTThe lipid aldehyde 4-oxo-2-nonenal (ONE) derived from peroxidation of n-6 polyunsaturated... more ABSTRACTThe lipid aldehyde 4-oxo-2-nonenal (ONE) derived from peroxidation of n-6 polyunsaturated fatty acids and generated in parallel to 4-hydroxynonenal (HNE) is a highly reactive protein crosslinker. Crosslinking of proteins in high-density lipoprotein (HDL) by lipid peroxidation products causes HDL dysfunction and contributes to atherogenesis. While HNE is relatively well studied, the relevance of ONE in atherosclerosis and in modifying HDL has not been examined. In the present study, we found a significant increase in ONE-ketoamide (lysine) adducts in HDL derived from patients with familial hypercholesterolemia (FH) (1620 ± 985.4 pmol/mg) compared to healthy controls (664 ± 219.5 pmol/mg). ONE crosslinked apoA-I on HDL at a concentration of >3 mol ONE per 10 mol apoA-I (0.3 eq), which is 100-fold lower than HNE but comparable to the potent protein crosslinker, isolevuglandin. ONE-modified HDL partially inhibited the ability of HDL to protect against LPS-induced TNFα and IL-...

The epidermis expresses cyclooxygenases, lipoxygenases and cytochromes P450, which utilize arachi... more The epidermis expresses cyclooxygenases, lipoxygenases and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12-and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-14 C]-arachidonic acid. The majority of the [ 14 C]products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) co-eluted with the CYP2B19 product 14,15-EET on a reversed-phase HPLC system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal [ 14 C]-products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPHdependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic This article has not been copyedited and formatted. The final version may differ from this version.

Alzheimer's & Dementia, 2008

Alzheimer's & Dementia, 2006

Protein …, 2006

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo... more Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification ...

Hypertension, 2020

Supplemental Digital Content is available in the text. Hypertension remains a major health proble... more Supplemental Digital Content is available in the text. Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochond...

The pathogenesis of atherosclerosis is accelerated by oxidative stress, which produces lipid pero... more The pathogenesis of atherosclerosis is accelerated by oxidative stress, which produces lipid peroxidation. Among the products of lipid peroxidation are highly reactive dicarbonyls that covalently modify proteins. We investigated the impact of treatment with the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in hyperlipidemic Ldlr-/- mice, a model of familial hypercholesterolemia (FH). Compared to mice treated with vehicle, 2-HOBA significantly decreased atherosclerosis in hypercholesterolemic Ldlr-/- mice by 31% in the proximal aortas and 60% in en face aortas, in the absence of changes in blood lipid levels. 2-HOBA reduced malondialdehyde (MDA) content in HDL, LDL, and in the atherosclerotic lesions. Consuming a Western diet increased plasma MDA-apoAI adduct levels in Ldlr-/- mice. 2-HOBA inhibited MDA-apoAI formation and increased the capacity of the mouse HDL to reduce macrophage cholesterol stores. Furthermore, 2-HOBA diminished oxidative...

Archives of Biochemistry and Biophysics, 2005

CYP2B19 is an arachidonic acid monooxygenase highly expressed in the outer, diVerentiated cell la... more CYP2B19 is an arachidonic acid monooxygenase highly expressed in the outer, diVerentiated cell layers of mouse epidermis. We aimed to establish whether CYP2B19 is the source of epidermal epoxyeicosatrienoic acids (EETs), which are implicated in mechanisms regulating epidermal corniWcation. We show that primary cultures of mouse epidermal keratinocytes expressed native CYP2B19, as determined by mass spectrometry. DiVerentiation upregulated CYP2B19 mRNA levels (t39-fold) detected by real-time PCR, CYP2B19 immunoreactivity detected by Western blotting, and cellular levels of the CYP2B19 product 11,12-EET. Cellular 11,12-EET formed from endogenous arachidonic acid increased preferentially (4-to 12-fold) at Day 4 or 5 of diVerentiation, compared with undiVerentiated (Day 0) keratinocyte cultures. Temporally, these results concur with the maximal levels of CYP2B19 mRNA measured at Day 2 and CYP2B19 immunoreactivity at Day 4. We conclude that while mouse epidermis likely expresses multiple cytochrome P450 enzymes, existing evidence supports native CYP2B19 as being the major source of epidermal EET formation.

Obesity Research & Clinical Practice

Alzheimer's & Dementia, 2006

Protein Science, 2006

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo... more Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.

Journal of Pharmacology and Experimental Therapeutics, 2005

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arach... more The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved th...

Journal of Biological Chemistry