Publications by authors named "Henriette Remmer"

14 Publications

  • Page 1 of 1

Valproic Acid Protects Against Acute Kidney Injury in Hemorrhage and Trauma.

J Surg Res 2021 May 20;266:222-229. Epub 2021 May 20.

Department of Surgery, University of Michigan, Ann Arbor, MI; Department of Surgery, Northwestern University, Chicago, IL.

Introduction: Trauma is the leading cause of death among young people. These patients have a high incidence of kidney injury, which independently increases the risk of mortality. As valproic acid (VPA) treatment has been shown to improve survival in animal models of lethal trauma, we hypothesized that it would also attenuate the degree of acute kidney injury.

Methods: We analyzed data from two separate experiments where swine were subjected to lethal insults.  Model 1: hemorrhage (50% blood volume hemorrhage followed by 72-h damage control resuscitation). Model 2: polytrauma (traumatic brain injury, 40% blood volume hemorrhage, femur fracture, rectus crush and grade V liver laceration). Animals were resuscitated with normal saline (NS) +/- VPA 150 mg/kg after a 1-h shock phase in both models (n = 5-6/group). Serum samples were analyzed for creatinine (Cr) using colorimetry on a Liasys 330 chemistry analyzer. Proteomic analysis was performed on kidney tissue sampled at the time of necropsy.

Results: VPA treatment significantly (P < 0.05) improved survival in both models. (Model 1: 80% vs 20%; Model 2: 83% vs. 17%). Model 1 (Hemorrhage alone): Cr increased from a baseline of 1.2 to 3.0 in NS control animals (P < 0.0001) 8 h after hemorrhage, whereas it rose only to 2.1 in VPA treated animals (P = 0.004). Model 2 (Polytrauma): Cr levels increased from baseline of 1.3 to 2.5 mg/dL (P = 0.01) in NS control animals 4 h after injury but rose to only 1.8 in VPA treated animals (P = 0.02). Proteomic analysis of kidney tissue identified metabolic pathways were most affected by VPA treatment.

Conclusions: A single dose of VPA (150 mg/kg) offers significant protection against acute kidney injury in swine models of polytrauma and hemorrhagic shock.
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http://dx.doi.org/10.1016/j.jss.2021.04.014DOI Listing
May 2021

Assessment of the Cytoprotective Effects of High-Dose Valproic Acid Compared to a Clinically Used Lower Dose.

J Surg Res 2021 May 11;266:125-141. Epub 2021 May 11.

Department of Surgery, University of Michigan Health System, Ann Arbor, Michigan; Department of Surgery, Feinberg School of Medicine/Northwestern University, Chicago, Illinois. Electronic address:

Objective: Valproic acid (VPA) treatment improves survival in animal models of injuries on doses higher than those allowed by Food and Drug Administration (FDA). We investigated the proteomic alterations induced by a single high-dose (140mg/kg) of VPA (VPA140) compared to the FDA-approved dose of 30mg/kg (VPA30) in healthy humans. We also describe the proteomic and transcriptomic changes induced by VPA140 in an injured patient. We hypothesized that VPA140 would induce cytoprotective changes in the study participants.

Methods: Serum samples were obtained from healthy subjects randomized to two groups; VPA140 and VPA30 at 3 timepoints: 0h(baseline), 2h, and 24h following infusion(n = 3/group). Samples were also obtained from an injured patient that received VPA140 at 0h, 6h and 24h following infusion. Proteomic analyses were performed using liquid chromatography-mass spectrometry (LC-MS/MS), and transcriptomic analysis was performed using RNA-sequencing. Differentially expressed (DE) proteins and genes were identified for functional annotation and pathway analysis using iPathwayGuide and gene set enrichment analysis (GSEA), respectively.

Results: For healthy individuals, a dose comparison was performed between VPA140 and VPA30 groups at 2 and 24 h. Functional annotation showed that top biological processes in VPA140 versus VPA30 analysis at 2 h included regulation of fatty acid (P = 0.002) and ATP biosynthesis (P = 0.007), response to hypoxia (P = 0.017), cell polarity regulation (P = 0.031), and sequestration of calcium ions (P = 0.031). Top processes at 24 h in VPA140 versus VPA30 analysis included amino acid metabolism (P = 0.023), collagen catabolism (P = 0.023), and regulation of protein breakdown (P = 0.023). In the injured patient, annotation of the DE proteins in the serum showed that top biological processes at 2 h included neutrophil chemotaxis (P = 0.002), regulation of cellular response to heat (P = 0.008), regulation of oxidative stress (P = 0.008) and regulation of apoptotic signaling pathway (P = 0.008). Top biological processes in the injured patient at 24 h included autophagy (P = 0.01), glycolysis (P = 0.01), regulation of apoptosis (P = 0.01) and neuron apoptotic processes (P = 0.02).

Conclusions: VPA140 induces cytoprotective changes in human proteome not observed in VPA30. These changes may be responsible for its protective effects in response to injuries.
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http://dx.doi.org/10.1016/j.jss.2021.03.025DOI Listing
May 2021

Valproic acid treatment rescues injured tissues after traumatic brain injury.

J Trauma Acute Care Surg 2020 12;89(6):1156-1165

From the Department of Surgery (B.E.B., L.P., A.I., A.A.S., A.Z.S., R.L.O., G.K.W., M.T.K., A.M.W., H.B.A.), Department of Biological Chemistry (H.A.R.), and Department of Clinical Pharmacy (M.P.P.), University of Michigan, Ann Arbor, Michigan.

Background: No agents that are specifically neuroprotective are currently approved to emergently treat patients with traumatic brain injury (TBI). The histone deacetylase inhibitor, high-dose valproic acid (VPA) has been shown to have cytoprotective potential in models of combined TBI and hemorrhagic shock, but it has not been tested in an isolated TBI model. We hypothesized that VPA, administered after isolated TBI, will penetrate the injured brain, attenuate the lesion size, and activate prosurvival pathways.

Methods: Yorkshire swine were subjected to severe TBI by cortical impact. One hour later, animals were randomized to VPA treatment (150 mg/kg delivered intravenously for 1 hour; n = 4) or control (saline vehicle; n = 4) groups. Seven hours after injury, animals were sacrificed, and brain lesion size was measured. Mass spectrometry imaging was used to visualize and quantitate brain tissue distribution of VPA. Sequential serum samples were assayed for key biomarkers and subjected to proteomic and pathway analysis.

Results: Brain lesion size was 50% smaller (p = 0.01) in the VPA-treated animals (3,837 ± 948 mm) compared with the controls (1,900 ± 614 mm). Endothelial regions had eightfold higher VPA concentrations than perivascular regions by mass spectrometry imaging, and it readily penetrated the injured brain tissues. Serum glial fibrillary acid protein was significantly lower in the VPA-treated compared with the control animals (p < 0.05). More than 500 proteins were differentially expressed in the brain, and pathway analysis revealed that VPA affected critical modulators of TBI response including calcium signaling pathways, mitochondria metabolism, and biosynthetic machinery.

Conclusion: Valproic acid penetrates injured brain tissues and exerts neuroprotective and prosurvival effects that resulted in a significant reduction in brain lesion size after isolated TBI. Levels of serum biomarkers reflect these changes, which could be useful for monitoring the response of TBI patients during clinical studies.
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http://dx.doi.org/10.1097/TA.0000000000002918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830776PMC
December 2020

Citrullinated Inhibitor of DNA Binding 1 Is a Novel Autoantigen in Rheumatoid Arthritis.

Arthritis Rheumatol 2019 08 21;71(8):1241-1251. Epub 2019 Jun 21.

University of Michigan Medical School, Ann Arbor.

Objective: To explore the intrinsic role of inhibitor of DNA binding 1 (ID-1) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and to investigate whether ID-1 is citrullinated and autoantigenic in RA.

Methods: RA patient serum ID-1 levels were measured before and after infliximab treatment. RA FLS were transfected with a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 construct targeting ID-1 to examine the effects of ID-1 deletion. RA synovial fluid (SF) and homogenized synovial tissue (ST) were immunoprecipitated for ID-1 and measured for citrullinated residues using an enzyme-linked immunosorbent assay and Western blotting. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on in vitro-citrullinated recombinant human ID-1 (cit-ID-1) to localize the sites of citrullination. Normal and RA sera and SF were analyzed by immunodot blotting for anti-citrullinated protein antibodies (ACPAs) to cit-ID-1.

Results: RA patient serum ID-1 levels positively correlated with several disease parameters and were reduced after infliximab treatment. RA FLS displayed reduced growth and a robust increase in interleukin-6 (IL-6) and IL-8 production upon deletion of ID-1. ID-1 immunodepletion significantly reduced the levels of citrullinated residues in RA SF, and citrullinated ID-1 was detected in homogenized RA ST (n = 5 samples; P < 0.05). Immunodot blot analyses revealed ACPAs to cit-ID-1 but not to native ID-1, in RA peripheral blood (PB) sera (n = 30 samples; P < 0.001) and SF (n = 18 samples; P < 0.05) but not in normal PB sera. Following analyses of LC-MS/MS results for citrullination sites and corresponding reactivity in immunodot assays, we determined the critical arginines in ID-1 for autoantigenicity: R33, R52, and R121.

Conclusion: Novel roles of ID-1 in RA include regulation of FLS proliferation and cytokine secretion as well as autoantigenicity following citrullination.
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http://dx.doi.org/10.1002/art.40886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663620PMC
August 2019

C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae.

PLoS Pathog 2018 10 23;14(10):e1007341. Epub 2018 Oct 23.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, United States of America.

Vibrio cholerae and a subset of other Gram-negative bacteria, including Acinetobacter baumannii, express proteins with a C-terminal tripartite domain called GlyGly-CTERM, which consists of a motif rich in glycines and serines, followed by a hydrophobic region and positively charged residues. Here we show that VesB, a V. cholerae serine protease, requires the GlyGly-CTERM domain, the intramembrane rhomboid-like protease rhombosortase, and the type II secretion system (T2SS) for localization at the cell surface. VesB is cleaved by rhombosortase to expose the second glycine residue of the GlyGly-CTERM motif, which is then conjugated to a glycerophosphoethanolamine-containing moiety prior to engagement with the T2SS and outer membrane translocation. In support of this, VesB accumulates intracellularly in the absence of the T2SS, and surface-associated VesB activity is no longer detected when the rhombosortase gene is inactivated. In turn, when VesB is expressed without an intact GlyGly-CTERM domain, VesB is released to the extracellular milieu by the T2SS and does not accumulate on the cell surface. Collectively, our findings suggest that the posttranslational modification of the GlyGly-CTERM domain is essential for cell surface localization of VesB and other proteins expressed with this tripartite extension.
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http://dx.doi.org/10.1371/journal.ppat.1007341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219818PMC
October 2018

Rapid valproic acid-induced modulation of the traumatic proteome in a porcine model of traumatic brain injury and hemorrhagic shock.

J Surg Res 2018 08 25;228:84-92. Epub 2018 Apr 25.

Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan. Electronic address:

Background: Histone deacetylase inhibitors such as valproic acid (VPA) improve survival in lethal models of hemorrhagic shock and polytrauma. Although VPA is known to modulate transcription, its ability to reduce mortality within minutes of administration suggests involvement of a rapid, posttranslational mechanism. We hypothesized that VPA treatment would cause proteomic changes within minutes of treatment including quantitative and/or posttranslational differences in structural and/or effector proteins.

Materials And Methods: We used a porcine model of traumatic brain injury (computer-controlled cortical impact, 12 mm depth) and hemorrhagic shock (40% hemorrhage). Animals were kept in shock for 2 h and randomized to two groups (n = 3): normal saline (volume = 3:1 hemorrhage volume) or normal saline + VPA (150 mg/kg, single dose). Peripheral blood mononuclear cells were collected at baseline, postshock, and postresuscitation. Intracellular protein profiles were assessed using 1 dimensional gel electrophoresis, liquid chromatography, mass spectrometry, and analyzed with Ingenuity Pathway Analysis software.

Results: Animals treated with VPA demonstrated significant proteomic changes. Quantitative differences were found in over 200 proteins including effector, regulatory, and structural proteins in critical cell signaling pathways. Posttranslational modification analysis demonstrated differential VPA-induced acetylation of lysine residues in histone and nonhistone proteins. Pathway analysis correlated these changes with significant increases in numerous prosurvival and cytoskeletal intracellular pathways, including Rho GTPase signaling (P = 1.66E-11), integrin signaling (P = 4.19E-21), and a decrease in Rho guanosine nucleotide dissociation inhibitor signaling (P = 4.83E-12).

Conclusions: In a porcine model of severe injuries, a single dose of VPA is associated with protective changes in the proteome that are measurable within minutes of treatment.
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http://dx.doi.org/10.1016/j.jss.2018.02.046DOI Listing
August 2018

Alterations in the human proteome following administration of valproic acid.

J Trauma Acute Care Surg 2016 12;81(6):1020-1027

From the Department of Surgery, University of Michigan, Ann Arbor, MI (P.E.G., I.H., V.N., T.B., C.T., B.L., T.L., H.B.A.); Department of Biological Chemistry, University of Michigan, Ann Arbor, MI (H.R.); and Department of Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor, MI (G.H., R.M.).

Background: High doses of the histone deacetylase inhibitor valproic acid (VPA, 150-400 mg/kg) improve outcomes in animal models of lethal insults. We are conducting a US Food and Drug Administration-approved Phase I, double-blind, placebo-controlled trial to evaluate the safety and tolerability of ascending doses of VPA in human volunteers. We hypothesized that VPA would induce significant changes in the proteome of healthy humans when given at doses lower than those used in prior animal studies.

Methods: Peripheral blood mononuclear cells were obtained from three healthy subjects randomized to receive VPA (120 mg/kg over 1 hour) at baseline and at 4 and 8 hours following infusion. Detailed proteomic analysis was performed using 1D gel electrophoresis, liquid chromatography, and mass spectrometry. Proteins with differential expression were chosen for functional annotation and pathway analysis using Ingenuity Pathway Analysis (Qiagen GmbH, Hilden, Germany) and Panther Gene Ontology.

Results: A total of 3,074 unique proteins were identified. The average number of proteins identified per sample was 1,716 ± 459. There were a total of 140 unique differentially expressed proteins (p < 0.05). There was a minor and inconsistent increase in histone and nonhistone protein acetylation. Functional annotation showed significant enrichment of apoptosis (p = 3.5E-43), cell death (p = 9.9E-72), proliferation of cells (p = 1.6E-40), dementia (p = 9.6E-40), amyloidosis (p = 6.3E-38), fatty acid metabolism (p = 4.6E-76), quantity of steroid (p = 4.2E-75), and cell movement (p = 1.9E-64).

Conclusions: Valproic acid induces significant changes to the proteome of healthy humans when given at a dose of 120 mg/kg. It alters the expression of key proteins and pathways, including those related to cell survival, without significant modification of protein acetylation. In the next part of the ongoing Phase I trial, we will study the effects of VPA on trauma patients in hemorrhagic shock.

Level Of Evidence: Therapeutic study, level V.
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http://dx.doi.org/10.1097/TA.0000000000001249DOI Listing
December 2016

Proteogenomic analysis of psoriasis reveals discordant and concordant changes in mRNA and protein abundance.

Genome Med 2015 4;7(1):86. Epub 2015 Aug 4.

Department of Dermatology, University of Michigan School of Medicine, Ann Arbor, MI 48109-2200 USA.

Background: Psoriasis is a chronic disease characterized by the development of scaly red skin lesions and possible co-morbid conditions. The psoriasis lesional skin transcriptome has been extensively investigated, but mRNA levels do not necessarily reflect protein abundance. The purpose of this study was therefore to compare differential expression patterns of mRNA and protein in psoriasis lesions.

Methods: Lesional (PP) and uninvolved (PN) skin samples from 14 patients were analyzed using high-throughput complementary DNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: We identified 4122 differentially expressed genes (DEGs) along with 748 differentially expressed proteins (DEPs). Global shifts in mRNA were modestly correlated with changes in protein abundance (r = 0.40). We identified similar numbers of increased and decreased DEGs, but 4-fold more increased than decreased DEPs. Ribosomal subunit and translation proteins were elevated within lesions, without a corresponding shift in mRNA expression (RPL3, RPS8, RPL11). We identified 209 differentially expressed genes/proteins (DEGPs) with corresponding trends at the transcriptome and proteome levels. Most DEGPs were similarly altered in at least one other skin disease. Psoriasis-specific and non-specific DEGPs had distinct cytokine-response patterns, with only the former showing disproportionate induction by IL-17A in cultured keratinocytes.

Conclusions: Our findings reveal global imbalance between the number of increased and decreased proteins in psoriasis lesions, consistent with heightened translation. This effect could not have been discerned from mRNA profiling data alone. High-confidence DEGPs were identified through transcriptome-proteome integration. By distinguishing between psoriasis-specific and non-specific DEGPs, our analysis uncovered new functional insights that would otherwise have been overlooked.
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http://dx.doi.org/10.1186/s13073-015-0208-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4527112PMC
August 2015

1,3-dinitrobenzene induces age- and region-specific oxidation to mitochondria-related proteins in brain.

Toxicol Sci 2015 May 24;145(1):48-58. Epub 2015 Feb 24.

*Toxicology Program, Department of Environmental Health Sciences, School of Public Health, Department of Biological Chemistry and Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109

Regions of the brain with high energy requirements are especially sensitive to perturbations in mitochondrial function. Hence, neurotoxicant exposures that target mitochondria in regions of high energy demand have the potential to accelerate mitochondrial damage inherently occurring during the aging process. 1,3-Dinitrobenzene (DNB) is a model neurotoxicant that selectively targets mitochondria in brainstem nuclei innervated by the eighth cranial nerve. This study investigates the role of age in the regional susceptibility of brain mitochondria-related proteins (MRPs) to oxidation following exposure to DNB. Male F344 rats (1 month old [young], 3 months old [adult], 18 months old [aged]) were exposed to 10 mg/kg DNB prior to mitochondrial isolation and histopathology experiments. Using a high-throughput proteomic approach, 3 important region- and age-related increases in DNB-induced MRP oxidation were determined: (1) brainstem mitochondria are ×3 more sensitive to DNB-induced oxidation than cortical mitochondria; (2) oxidation of brainstem MRPs is significantly higher than in cortical counterparts; and (3) MRPs from the brainstems of older rats are significantly more oxidized than those from young or adult rats. Furthermore, lower levels of DNB cause signs of intoxication (ataxia, chromodacryorrhea) and vacuolation of the susceptible neuropil in aged animals, while neither is observed in DNB-exposed young rats. Additionally, methemoglobin levels increase significantly in DNB-exposed adult and aged animals, but not young DNB-exposed animals. This suggests that oxidation of key MRPs observed in brainstem of aged animals is necessary for DNB-induced signs of intoxication and lesion formation. These results provide compelling evidence that environmental chemicals such as DNB may aid in the acceleration of injury to specific brain regions by inducing oxidation of sensitive mitochondrial proteins.
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http://dx.doi.org/10.1093/toxsci/kfv015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408960PMC
May 2015

Solar ultraviolet irradiation induces decorin degradation in human skin likely via neutrophil elastase.

PLoS One 2013 30;8(8):e72563. Epub 2013 Aug 30.

Department of Dermatology, University of Michigan, Ann Arbor, Michigan, United States of America.

Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072563PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758340PMC
April 2014

Disparate mechanisms of sICAM-1 production in the peripheral lung: contrast between alveolar epithelial cells and pulmonary microvascular endothelial cells.

Am J Physiol Lung Cell Mol Physiol 2008 Apr 15;294(4):L807-14. Epub 2008 Feb 15.

Pulmonary Section , Veterans Affairs Medical Center, Ann Arbor, MI 48105, USA.

Membrane-associated intercellular adhesion molecule-1 (mICAM-1; CD54) is constitutively expressed on the surface of type I alveolar epithelial cells (AEC). Soluble ICAM-1 (sICAM-1) may be produced by proteolytic cleavage of mICAM-1 or by alternative splicing of ICAM-1 mRNA. In contrast to inducible expression seen in most cell types, sICAM-1 is constitutively released by type I AEC and is present in normal alveolar lining fluid. Therefore, we compared the mechanism of sICAM-1 production in primary cultures of two closely juxtaposed cells in the alveolar wall, AEC and pulmonary microvascular endothelial cells (PVEC). AEC, but not PVEC, demonstrated high-level baseline expression of sICAM-1. Stimulation of AEC with TNFalpha or LPS resulted in minimal increase in AEC sICAM-1, whereas PVEC sICAM-1 was briskly induced in response to these signals. AEC sICAM-1 shedding was significantly reduced by treatment with a serine protease inhibitor, but not by cysteine, metalloprotease, or aspartic protease inhibitors. In contrast, none of these inhibitors effected sICAM-1 expression in PVEC. RT-PCR, followed by gel analysis of total RNA, suggests that alternatively spliced fragments are present in both cell types. However, a 16-mer oligopeptide corresponding to the juxtamembrane region of mICAM-1 completely abrogated sICAM-1 shedding in AEC but reduced stimulated PVEC sICAM-1 release by only 20%. Based on these data, we conclude that the predominant mechanism of sICAM-1 production likely differs in the two cell types from opposite sides of the alveolar wall.
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http://dx.doi.org/10.1152/ajplung.00398.2007DOI Listing
April 2008

A new alpha-galactosyl-binding protein from the mushroom Lyophyllum decastes.

Arch Biochem Biophys 2007 Nov 30;467(2):268-74. Epub 2007 Aug 30.

Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, USA.

A new alpha-galactosyl binding lectin was isolated from the fruiting bodies of the mushroom Lyopyllum decastes. It is a homodimer composed of noncovalently-associated monomers of molecular mass 10,276Da. The lectin's amino acid sequence was determined by cloning from a cDNA library using partial sequences determined by automated Edman sequencing and by mass spectrometry of enzyme-derived peptides. The sequence shows no significant homology to any known protein sequence. Analysis of carbohydrate binding specificity by a variety of approaches including precipitation with glycoconjugates and microcalorimetric titration reveals specificity towards galabiose (Gal alpha1,4Gal), a relatively rare disaccharide in humans. The lectin shares carbohydrate binding preference with the Shiga-like toxin, also known as verocytoxin, present in the bacteria Shigella dysenteriae and Escherichia. coli 0157:H7, both of which are causes of outbreaks of sometimes fatal food-borne illnesses.
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http://dx.doi.org/10.1016/j.abb.2007.08.017DOI Listing
November 2007

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban.

Biopolymers 2007 ;88(1):29-35

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

Electron paramagnetic resonance (EPR) was used to optimize the solid-phase peptide synthesis of a membrane-bound peptide labeled with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid). The incorporation of this paramagnetic amino acid results in a nitroxide spin label coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by EPR. We applied this approach to phospholamban, which regulates cardiac calcium transport. The synthesis of this amphipathic 52-amino-acid membrane peptide including TOAC is a challenge, especially in the addition of TOAC and the next several amino acids. Therefore, EPR of synthetic intermediates, reconstituted into lipid bilayers, was used to ensure complete coupling and 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. The attachment of Fmoc-TOAC-OH leads to strong immobilization of the spin label, whereas Fmoc deprotection dramatically mobilizes it, producing an EPR spectral peak that is completely resolved from that observed before deprotection. Similarly, coupling of the next amino acid (Ser) restores the spin label to strong immobilization, giving a peak that is completely resolved from that of the preceding step. For several subsequent steps, the effect of coupling and deprotection is similar but less dramatic. Thus, the sensitivity and resolution of EPR provides a quantitative monitor of completion at each of these critical steps in peptide synthesis. Mass spectrometry, circular dichroism, and Edman degradation were used in concert with EPR to verify the chemistry and characterize the secondary structure. In conclusion, the application of conventional analytical methods in combination with EPR offers an improved approach to optimize the accurate synthesis of TOAC spin-labeled membrane peptides.
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http://dx.doi.org/10.1002/bip.20618DOI Listing
March 2007

Cyclic and hairpin peptide complexes of heme.

J Am Chem Soc 2002 Oct;124(42):12394-5

School of Chemical Sciences, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.

We have synthesized and characterized a new class of heme-peptide complexes using disulfide-linked hairpin-turn and cyclic peptides and compared these to their linear analogues. The binding affinities, helicities, and mechanism of binding of linear, hairpin, and cyclic peptides to [FeIII(coproporphyrin-I)]+ have been determined. In a minimalist approach, we utilize amphiphilic peptide sequences (15-mers), where a central histidine provides heme ligation, and the hydrophobic effect is used to optimize heme-peptide complex stability. We have incorporated disulfide bridges between amphiphilic peptides to make hairpin and even cyclic peptides that bind heme extremely well, roughly 5 x 106 times more strongly than histidine itself. CD studies show that the cyclic peptide heme complexes are completely alpha-helical. NMR spectra of paramagnetic complexes of the peptides show that the 15-mer peptides bind sequentially, with an observable monopeptide, high-spin intermediate. In contrast, the cyclic peptide complexes ligate both imidazoles cooperatively to the heme, producing only a low-spin complex. Electrochemical measurements of the E1/2 of the FeIII(coproporphyrin-I)+ complexes of these peptides are all at fairly low potentials, ranging from -215 to -252 mV versus NHE at pH 7.
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http://dx.doi.org/10.1021/ja020912wDOI Listing
October 2002