Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry

The Ca²⁺/calmodulin activated phosphatase, calcineurin, is inactivated by H₂O₂ or superoxide-induced oxidation, both <i>in vivo</i> and <i>in vitro</i>. However, the potential for global and/or local conformation changes occurring within calcineurin as a function of oxidative modification, that may play a role in the inactivation process, has not been examined. Here, the susceptibility of calcineurin methionine residues toward H₂O₂-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC–electrospray ionization mass spectrometry and tandem mass spectrometry analysis. Then, regions within the protein complex that underwent significant conformational perturbation upon oxidative modification were identified by monitoring changes in the modification rates of accessible lysine residues between native and oxidized forms of calcineurin, using an amine-specific covalent labeling reagent, <i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and tandem mass spectrometry. Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca²⁺/CaM binding regulated calcineurin stimulation. These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca²⁺ binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center

Comprehensive Lipidome Profiling of Isogenic Primary and Metastatic Colon Adenocarcinoma Cell Lines

A “shotgun” lipidomics strategy consisting of sequential functional group selective chemical modification reactions coupled with high-resolution/accurate mass spectrometry and “targeted” tandem mass spectrometry (MS/MS) analysis has been developed and applied toward the comprehensive identification, characterization and quantitative analysis of changes in relative abundances of >600 individual glycerophospholipid, glycerolipid, sphingolipid and sterol lipids between a primary colorectal cancer (CRC) cell line, SW480, and its isogenic lymph node metastasized derivative, SW620. Selective chemical derivatization of glycerophosphoethanolamine and glycerophosphoserine lipids using a “fixed charge” sulfonium ion containing, d₆-<i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (d₆-DMBNHS) reagent was used to eliminate the possibility of isobaric mass overlap of these species with the precursor ions of all other lipids in the crude extracts, thereby enabling their unambiguous assignment, while subsequent selective mild acid hydrolysis of plasmenyl (vinyl-ether) containing lipids using formic acid enabled these species to be readily differentiated from isobaric mass plasmanyl (alkyl-ether) containing lipids. Using this approach, statistically significant differences in the abundances of numerous lipid species previously identified as being associated with cancer progression or that play known roles as mediators in a range of physiological and pathological processes were observed between the SW480 and SW620 cells. Most notably, these included increased plasmanylcholine and triglyceride lipid levels, decreased plasmenylethanolamine lipids, decreased C-16 containing sphingomyelin and ceramide lipid levels, and a dramatic increase in the abundances of total cholesterol ester and triglyceride lipids in the SW620 cells compared to those in the SW480 cells

Comprehensive Lipidome Profiling of Isogenic Primary and Metastatic Colon Adenocarcinoma Cell Lines

A “shotgun” lipidomics strategy consisting of sequential functional group selective chemical modification reactions coupled with high-resolution/accurate mass spectrometry and “targeted” tandem mass spectrometry (MS/MS) analysis has been developed and applied toward the comprehensive identification, characterization and quantitative analysis of changes in relative abundances of >600 individual glycerophospholipid, glycerolipid, sphingolipid and sterol lipids between a primary colorectal cancer (CRC) cell line, SW480, and its isogenic lymph node metastasized derivative, SW620. Selective chemical derivatization of glycerophosphoethanolamine and glycerophosphoserine lipids using a “fixed charge” sulfonium ion containing, d₆-<i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (d₆-DMBNHS) reagent was used to eliminate the possibility of isobaric mass overlap of these species with the precursor ions of all other lipids in the crude extracts, thereby enabling their unambiguous assignment, while subsequent selective mild acid hydrolysis of plasmenyl (vinyl-ether) containing lipids using formic acid enabled these species to be readily differentiated from isobaric mass plasmanyl (alkyl-ether) containing lipids. Using this approach, statistically significant differences in the abundances of numerous lipid species previously identified as being associated with cancer progression or that play known roles as mediators in a range of physiological and pathological processes were observed between the SW480 and SW620 cells. Most notably, these included increased plasmanylcholine and triglyceride lipid levels, decreased plasmenylethanolamine lipids, decreased C-16 containing sphingomyelin and ceramide lipid levels, and a dramatic increase in the abundances of total cholesterol ester and triglyceride lipids in the SW620 cells compared to those in the SW480 cells

Synthesis, Structural Characterization, and Gas-Phase Unimolecular Reactivity of Bis(diphenylphosphino)amino Copper Hydride Nanoclusters [Cu₃(X)(μ₃‑H)((PPh₂)₂NH)₃](BF₄), Where X = μ₂‑Cl and μ₃‑BH₄

An electrospray ionization mass spectrometry (ESI-MS) survey of the types of cationic copper clusters formed from an acetonitrile solution containing a 1:1:20 mixture of tetrakis­(acetonitrile)­copper­(I) tetrafluoroborate [Cu­(MeCN)₄(BF₄)], bis­(diphenylphosphino)­amine (dppa = (Ph₂P)₂NH = L), and NaBH₄ revealed a major peak, which based on both the accurate masses and isotope distribution was assigned as [Cu₃(BH₄)­(H)­(L)₃]⁺. This prompted synthetic efforts resulting in isolation of the dppa ligated trinuclear copper hydride nanoclusters, [Cu₃(μ₂-Cl)­(μ₃-H)­(L)₃]­(BF₄) and [Cu₃(μ₃-BH₄)­(μ₃-H)­(L)₃]­(BF₄), which were subsequently structurally characterized using high resolution ESI-MS, X-ray crystallography, NMR, and IR spectroscopy. The X-ray structures reveal a common structural feature of the cation, in which the three copper­(I) ions adopt a planar trinuclear Cu₃ geometry coordinated on the bottom face by a μ₃-hydride and surrounded by three dppa ligands. ESI-MS of [Cu₃(<i>μ</i>₂-Cl)­(μ₃-H)­(L)₃]­(BF₄) and [Cu₃(μ₃-BH₄)­(μ₃-H)­(L)₃]­(BF₄) produces [Cu₃(μ₂-Cl)­(μ₃-H)­(L)₃]⁺ and [Cu₃(μ₃-BH₄)­(μ₃-H)­(L)₃]⁺. The unimolecular gas-phase ion chemistry of these cations was examined under multistage tandem mass spectrometry conditions using collision-induced dissociation (CID). CID of both cations proceeds via ligand loss to give [Cu₃(μ₃-H)­(X)­(L)₂]⁺, which is in competition with BH₃ loss in the case of X = BH₄. DFT calculations on the fragmentation of [Cu₃(μ₃-BH₄)­(μ₃-H)­(L^Me)₃]⁺ suggest that BH₃ loss produces the hitherto elusive [Cu₃(μ₃-H)­(μ₂<i>-</i>H)­(L^Me)₃]⁺, which undergoes further fragmentation via H₂ loss. CID of the deuterium labeled cluster [Cu₃(μ₃-D)­(μ₃-BD₄)­(L)₃]⁺ reveals that the competing losses of ligand and BD₃ yield [Cu₃(μ₃-BD₄)­(μ₃-D)­(L)₂]⁺ and [Cu₃(D)₂(L)₃]⁺ as primary products, which subsequently fragment via further losses of BD₃ or a ligand to give [Cu₃(D)₂(L)₂]⁺. The coordinated hydrides in the latter ion are activated toward elimination of D₂ to give [Cu₃(L)₂]⁺. Loss of HD and 2HD are minor channels, consistent with higher DFT predicted endothermicities to form [Cu₃(D)­(L)­(L-H)]⁺ and [Cu₃(L-H)₂]⁺

Altered Lipid Metabolism in Residual White Adipose Tissues of Bscl2 Deficient Mice

<div><p>Mutations in BSCL2 underlie human congenital generalized lipodystrophy type 2 disease. We previously reported that <i>Bscl2</i><b>^−/−</b> mice develop lipodystrophy of white adipose tissue (WAT) due to unbridled lipolysis. The residual epididymal WAT (EWAT) displays a browning phenotype with much smaller lipid droplets (LD) and higher expression of brown adipose tissue marker proteins. Here we used targeted lipidomics and gene expression profiling to analyze lipid profiles as well as genes involved in lipid metabolism in WAT of wild-type and <i>Bscl2^−/−</i> mice. Analysis of total saponified fatty acids revealed that the residual EWAT of <i>Bscl2^−/−</i> mice contained a much higher proportion of oleic_18:1n9 acid concomitant with a lower proportion of palmitic_16:0 acid, as well as increased n3- polyunsaturated fatty acids (PUFA) remodeling. The acyl chains in major species of triacylglyceride (TG) and diacylglyceride (DG) in the residual EWAT of <i>Bscl2^−/−</i> mice were also enriched with dietary fatty acids. These changes could be reflected by upregulation of several fatty acid elongases and desaturases. Meanwhile, <i>Bscl2^−/−</i> adipocytes from EWAT had increased gene expression in lipid uptake and TG synthesis but not de novo lipogenesis. Both mitochondria and peroxisomal β-oxidation genes were also markedly increased in <i>Bscl2^−/−</i> adipocytes, highlighting that these machineries were accelerated to shunt the lipolysis liberated fatty acids through uncoupling to dissipate energy. The residual subcutaneous white adipose tissue (ScWAT) was not browning but displays similar changes in lipid metabolism. Overall, our data emphasize that, other than being essential for adipocyte differentiation, Bscl2 is also important in fatty acid remodeling and energy homeostasis.</p></div

De novo lipogenesis and metabolic pathways of MUFA and PUFAs.

<p>Fatty acids are synthesized through de novo lipogenesis (DNL) or converted from dietary palmitic_16:0, oleic_18:1n9, linoleic_18:2n6 and α-linolenic_18:3n3 acids to long chain unsaturated fatty acids <i>in vivo</i> by a series of elongation by elongases (Elovl) and desaturation (Δ5 desaturase (Δ5D/Fasd1), Δ6 desaturase (Δ6D/Fads2), or Δ9-desaturase (Δ9D/Scd1)). Fatty acids that accumulate in animal and human tissues are in solid boxes. Fatty acids derived from normal rodent chow diet are shaded in gray.</p

The residual <i>Bscl2</i><i>⁻</i>^{<i>/</i><i>−</i>} subcutaneous white adipose tissues were not browning but had similar altered lipid metabolism.

<p>qPCR analyses of BAT specific genes Ucp1 and Elovl3, lipolytic product activated transcription factor Pparα and its targeted genes Cpt1α and Acox2 (A); and genes involved in elongation, desaturation and TG synthesis (B) in isolated adipocytes from ScWAT of <i>Bscl2^+/+</i> and <i>Bscl2^−/−</i> mice. Each sample was pooled from 3-4 6-week-old nonfasting male wild-type and <i>Bscl2</i>^−/− mice (<i>n</i>  =  4–5). *: P<0.05; **: p<0.005. (C) TLC analysis of total lipids extracted from ScWAT of male non-fasting <i>Bscl2^+/+ and Bscl2^−/−</i> mice (n = 5–6). Total lipids from equal amounts of tissue for each genotype were loaded.</p

Lipidomic analysis of DGs by shotgun mass spectrometry of EWAT from <i>Bscl2^+/+</i> and <i>Bscl2^−/−</i> mice.

<p>DG species were determined using high resolution ESI-MS and confirmed via product ion scan mode CID-MS/MS as described in Methods (n = 3 pooled from 6 animals). Data are expressed as % total DG ion abundance in each genotype. Data are presented as means ± SD. *: p<0.05; **: p<0.005. Arrows indicate upregulation or downregulation vs. <i>Bscl^+/+</i> EWAT.</p

Altered fatty acid compositions suggest increased rate of fatty acid mobilization in residual <i>Bscl2^−/−</i> EWAT.

<p>A) Identification and quantification of changes in total adipose tissue saponified fatty acids by RP-HPLC. B) Ratio of oleic_18:1n9/palmitic_16:0 acids. C) End product/precursor ratio of DHA_22:6n3/α-linolenic_18:3n3 acids. D) Unsaturation index based on the number of double bonds per fatty acyl residue. n = 3 with each sample pooled from EWAT fat pads from 2 animals. *: p<0.05; **: p<0.005.</p

Lipidomic analysis of TGs by shotgun mass spectrometry of EWAT from <i>Bscl2^+/+</i> and <i>Bscl2^−/−</i> mice.

<p>TG species were determined using high resolution ESI-MS and confirmed via product ion scan mode CID-MS/MS and –MS³ as described in Methods (n = 3 pooled from 6 animals). Data are expressed as % total TG ion abundance in each genotype. Only the 46 TG species observed at >0.1% total TG ion abundance in <i>Bscl2^+/+</i> EWAT are listed. Data are presented as means ± SD. *: p<0.05; **: p<0.005. Arrows indicate upregulation or downregulation vs. <i>Bscl^+/+</i> EWAT.</p