Publications by authors named "Hans J Vogel"

245 Publications

An Integrative Approach to Determine 3D Protein Structures Using Sparse Paramagnetic NMR Data and Physical Modeling.

Front Mol Biosci 2021 12;8:676268. Epub 2021 Aug 12.

Department of Chemistry, University of Calgary, Calgary, AB, Canada.

Paramagnetic nuclear magnetic resonance (NMR) methods have emerged as powerful tools for structure determination of large, sparsely protonated proteins. However traditional applications face several challenges, including a need for large datasets to offset the sparsity of restraints, the difficulty in accounting for the conformational heterogeneity of the spin-label, and noisy experimental data. Here we propose an integrative approach to structure determination combining sparse paramagnetic NMR with physical modelling to infer approximate protein structural ensembles. We use calmodulin in complex with the smooth muscle myosin light chain kinase peptide as a model system. Despite acquiring data from samples labeled only at the backbone amide positions, we are able to produce an ensemble with an average RMSD of ∼2.8 Å from a reference X-ray crystal structure. Our approach requires only backbone chemical shifts and measurements of the paramagnetic relaxation enhancement and residual dipolar couplings that can be obtained from sparsely labeled samples.
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http://dx.doi.org/10.3389/fmolb.2021.676268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8407082PMC
August 2021

Obituary.

Authors:
Hans J Vogel

Biochim Biophys Acta Biomembr 2021 Aug 27;1863(12):183720. Epub 2021 Aug 27.

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http://dx.doi.org/10.1016/j.bbamem.2021.183720DOI Listing
August 2021

Preface for the BBA Biomembranes Special Issue on "Mechanisms of function and regulation of large transport membrane channels: Roles of complex membrane processes".

Biochim Biophys Acta Biomembr 2021 Nov 29;1863(11):183717. Epub 2021 Jul 29.

University of Calgary, Department of Biological Sciences, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.bbamem.2021.183717DOI Listing
November 2021

Seminal plasma metabolomics reveals lysine and serine dysregulation as unique features distinguishing between prostate cancer tumors of Gleason grades 6 and 7.

Prostate 2021 Aug 7;81(11):713-720. Epub 2021 Jun 7.

Department of Surgery, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

Background: Prostate cancer (PCa) is a metabolic disease. Most men are diagnosed with low grade indolent disease and differentiating these men from those who have life threatening cancer is a challenging but important clinical dilemma. There are currently limited biomarkers that can distinguish between the indolent Gleason grade 6 and higher-grade disease. Moreover, some individuals initially diagnosed with low grade disease progress to higher grade disease. Currently prostate biopsies are the only reliable methods of stratifying risk, but biopsies can cause significant morbidity, sample only a small portion of the gland and are costly. Therefore, biomarkers distinguishing between indolent and aggressive patterns of PCa are urgently required to minimize biopsy-associated morbidity, prevent over-treatment of indolent PCa and to better stratify patients for appropriate treatment.

Methods: Seminal fluid samples were collected from normal individuals (n = 13) Before infertility treatment and histologically confirmed PCa patients (n = 51). H Nuclear magnetic resonance spectroscopy and orthogonal partial least square discriminant analysis were used to compare the populations.

Results: Alterations in amino acids levels, specifically lysine and serine and changes in glycolytic intermediates were the most significant metabolic features associated with differences between healthy controls and PCa and between Gleason grade 6 (GS6) and Gleason grade 7 (GS7) samples. Orthogonal partial least square plots discriminated healthy controls from PCa samples (R  = 0.54, Q  = 0.31; area under the receiver operating characteristics curve [AUC] = 0.96), and GS6 from GS7 samples (R  = 0.62, Q  = 0.49; AUC = 0.98) based on lysine and serine content.

Conclusion: This study suggests that seminal plasma metabolomics profiling of seminal fluid is a promising means of differentiating indolent from aggressive disease. Particularly, lysine and serine levels may be able to differentiate GS6 from GS7 disease.
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http://dx.doi.org/10.1002/pros.24145DOI Listing
August 2021

ARDS metabolic fingerprints: characterization, benchmarking, and potential mechanistic interpretation.

Am J Physiol Lung Cell Mol Physiol 2021 07 5;321(1):L79-L90. Epub 2021 May 5.

Department of Critical Care Medicine, University of Calgary, Calgary, Alberta, Canada.

In this study, we aimed to identify acute respiratory distress syndrome (ARDS) metabolic fingerprints in selected patient cohorts and compare the metabolic profiles of direct versus indirect ARDS and hypoinflammatory versus hyperinflammatory ARDS. We hypothesized that the biological and inflammatory processes in ARDS would manifest as unique metabolomic fingerprints that set ARDS apart from other intensive care unit (ICU) conditions and could help examine ARDS subphenotypes and clinical subgroups. Patients with ARDS ( = 108) and ICU ventilated controls ( = 27) were included. Samples were randomly divided into 2/3 training and 1/3 test sets. Samples were analyzed using H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Twelve proteins/cytokines were also measured. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to select the most differentiating ARDS metabolites and protein/cytokines. Predictive performance of OPLS-DA models was measured in the test set. Temporal changes of metabolites were examined as patients progressed through ARDS until clinical recovery. Metabolic profiles of direct versus indirect ARDS subgroups and hypoinflammatory versus hyperinflammatory ARDS subgroups were compared. Serum metabolomics and proteins/cytokines had similar area under receiver operator curves when distinguishing ARDS from ICU controls. Pathway analysis of ARDS differentiating metabolites identified a dominant involvement of serine-glycine metabolism. In longitudinal tracking, the identified pathway metabolites generally exhibited correction by 7-14 days, coinciding with clinical improvement. ARDS subphenotypes and clinical subgroups were metabolically distinct. However, our identified metabolic fingerprints are not ARDS diagnostic biomarkers, and further research is required to ascertain generalizability. In conclusion, patients with ARDS are metabolically different from ICU controls. ARDS subphenotypes and clinical subgroups are metabolically distinct.
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http://dx.doi.org/10.1152/ajplung.00077.2021DOI Listing
July 2021

Harnessing the Benefits of Neuroinflammation: Generation of Macrophages/Microglia with Prominent Remyelinating Properties.

J Neurosci 2021 04 12;41(15):3366-3385. Epub 2021 Mar 12.

Hotchkiss Brain Institute and Department of Clinical Neurosciences

Excessive inflammation within the CNS is injurious, but an immune response is also required for regeneration. Macrophages and microglia adopt different properties depending on their microenvironment, and exposure to IL4 and IL13 has been used to elicit repair. Unexpectedly, while LPS-exposed macrophages and microglia killed neural cells in culture, the addition of LPS to IL4/IL13-treated macrophages and microglia profoundly elevated IL10, repair metabolites, heparin binding epidermal growth factor trophic factor, antioxidants, and matrix-remodeling proteases. In C57BL/6 female mice, the generation of M(LPS/IL4/IL13) macrophages required TLR4 and MyD88 signaling, downstream activation of phosphatidylinositol-3 kinase/mTOR and MAP kinases, and convergence on phospho-CREB, STAT6, and NFE2. Following mouse spinal cord demyelination, local LPS/IL4/IL13 deposition markedly increased lesional phagocytic macrophages/microglia, lactate and heparin binding epidermal growth factor, matrix remodeling, oligodendrogenesis, and remyelination. Our data show that a prominent reparative state of macrophages/microglia is generated by the unexpected integration of pro- and anti-inflammatory activation cues. The results have translational potential, as the LPS/IL4/IL13 mixture could be locally applied to a focal CNS injury to enhance neural regeneration and recovery. The combination of LPS and regulatory IL4 and IL13 signaling in macrophages and microglia produces a previously unknown and particularly reparative phenotype devoid of pro-inflammatory neurotoxic features. The local administration of LPS/IL4/IL13 into spinal cord lesion elicits profound oligodendrogenesis and remyelination. The careful use of LPS and IL4/IL13 mixture could harness the known benefits of neuroinflammation to enable repair in neurologic insults.
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http://dx.doi.org/10.1523/JNEUROSCI.1948-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051677PMC
April 2021

Lactoferrin extends its reach into South America.

Biochem Cell Biol 2021 02;99(1):v-vii

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.

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http://dx.doi.org/10.1139/bcb-2021-0025DOI Listing
February 2021

Multimodal peripheral fluid biomarker analysis in clinically isolated syndrome and early multiple sclerosis.

Mult Scler Relat Disord 2021 May 3;50:102809. Epub 2021 Feb 3.

Departments of Clinical Neurosciences, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

Background: Increasing evidence suggests that various inflammatory, immunological and metabolic pathways are altered in the clinically isolated syndrome (CIS) of multiple sclerosis (MS). Moreover, recent diagnostic criteria have made possible the very early diagnosis of MS. We evaluated multiple fluid biomarkers in people with early MS and CIS.

Methods: We measured blood levels of cytokines, matrix metalloproteinases (MMPs), serum metabolomics and immune cell immunophenotyping in participants in the Trial of Minocycline in a Clinically Isolated Syndrome of Multiple Sclerosis.

Results: When compared with healthy controls, people with early MS/CIS had higher levels of eotaxin, MCP-3, IL-1 receptor antagonist, IL-1β, IL-9 and IP-10, as well as MMPs 1, 8 and 9. In metabolomics analysis, the alanine, aspartate and glutamate metabolism and the synthesis and degradation of ketone bodies pathways were altered compared to healthy controls. There were no differences in lymphocyte subpopulation numbers. Out of all these biomarkers, only MMP-1 was able to differentiate between early MS and CIS, and was found to correlate with lesion volume and gadolinium enhancing lesions on MRI.

Conclusion: The immunological and metabolic profile of CIS and early MS is remarkably similar, supporting that these are a continuum of a common underlying pathophysiological process.
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http://dx.doi.org/10.1016/j.msard.2021.102809DOI Listing
May 2021

Maternal and Cord Blood Metabolite Associations with Gestational Weight Gain and Pregnancy Health Outcomes.

J Proteome Res 2021 03 2;20(3):1630-1638. Epub 2021 Feb 2.

School of Human Kinetics, Faculty of Health Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.

Pre-pregnancy obesity and excessive gestational weight gain (GWG) are risk factors for future maternal and childhood obesity. Maternal obesity is potentially communicated to the fetus in part by the metabolome, altering the child's metabolic program in early development. Fasting maternal blood samples from 37 singleton pregnancies at 25-28 weeks of gestation were obtained from mothers with pre-pregnancy body mass indexes (BMIs) between 18 and 40 kg/m. Various health measures including GWG, diet, and physical activity were also assessed. At term (37-42 weeks), a venous umbilical cord sample was obtained. Serum metabolomic profiles were measured using nuclear magnetic resonance spectroscopy as well as a gut and metabolic hormone panel. Maternal and cord serum metabolites were tested for associations with pre-pregnancy BMI, GWG, health outcomes, and gut and metabolic hormones. While cord blood metabolites showed no significant correlation to maternal obesity status or other measured health outcomes, maternal serum metabolites showed distinct profiles for lean, overweight, and obese women. Additionally, four serum metabolites, namely, glutamate, lysine, pyruvate, and valine, allowed prediction of excessive GWG when pre-pregnancy BMI was controlled. Metabolic biomarkers predictive of GWG are reported and, if validated, could aid in the guidance of prenatal weight management plans as the majority of pregnancy weight gain occurs in the third trimester.
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http://dx.doi.org/10.1021/acs.jproteome.0c00854DOI Listing
March 2021

Caffeine-Containing Energy Shots Cause Acute Impaired Glucoregulation in Adolescents.

Nutrients 2020 Dec 16;12(12). Epub 2020 Dec 16.

College of Food, Agricultural and Environmental Sciences, The Ohio State University, Columbus, OH 43210, USA.

Caffeine-containing, nutritionally fortified energy shots are consumed at high rates by adolescents, yet little is known about their metabolic impact. The purpose of this study was to examine the consequences of small format, caffeinated energy shots on glucose metabolism and gastrointestinal hormone secretion in adolescents. Twenty participants aged 13-19 years participated in a double-blind, randomized cross-over study consisting of two trials separated by 1-4 weeks. Participants consumed a volume-matched caffeinated energy shot (CAF, 5 mg/kg) or a decaffeinated energy shot (DECAF) followed by a 2 h oral glucose tolerance test. Blood samples were collected and area under the curve (AUC) calculated for glucose, insulin and gut and metabolic hormones. Consumption of CAF resulted in a 25% increase in glucose and a 26% increase in insulin area under the curve (AUC, = 0.037; < 0.0001) compared to DECAF. No impact on gut hormones was observed. To further characterize responses, individuals were classified as either slow or fast caffeine metabolizers based on an allele score. Glucose intolerance was greater in genetically fast vs. slow caffeine metabolizers and differences between groups were supported by distinct serum metabolomics separation. Consumption of caffeine-containing energy shots results in acute impaired glucoregulation in healthy adolescents as characterized by hyperinsulinemia following an oral glucose challenge.
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http://dx.doi.org/10.3390/nu12123850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766305PMC
December 2020

Antibiofilm activity of lactoferrin-derived synthetic peptides against PAO1.

Biochem Cell Biol 2021 02 1;99(1):138-148. Epub 2020 Sep 1.

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.

Many pathogenic bacteria can protect themselves from the effects of antibiotics and the host immune response system by forming biofilms. Biofilms are polymer-entrapped bacterial cells, which adhere to each other and are often attached to a surface. Eradication of bacterial biofilms typically requires much higher concentrations of antibiotics than are normally needed to kill cultured planktonic cells, raising serious clinical concerns. In an attempt to prevent the formation of biofilms or to break up existing biofilms of pathogenic bacteria, herein we have used the standard crystal violet assay as well as the Calgary biofilm device to test several lactoferrin- and lactoferricin-derived antimicrobial peptides for their antibiofilm activity against PAO1. Our results revealed that the short bovine lactoferricin-derived RRWQWR-NH (20-25) hexapeptide has no activity against PAO1. Moreover, the longer human lactoferricin-derived peptide GRRRRSVQWCA (1-11) and the bovine lactoferrampin (268-284) peptide were also almost devoid of activity. However, several different "mix-and-match" dimeric versions of the two lactoferricin-derived peptides proved quite effective in preventing the formation of biofilms at low concentrations, and in some cases, could even eradicate an existing biofilm. Moreover, the full-length bovine lactoferricinB (17-41) peptide also displayed considerable antimicrobial activity. Some of the longer lactoferricin-derived dimeric peptides acted through a bactericidal mechanism, whereas others seemed to interfere in cell-signalling processes. Taken together, our results indicate that synthetic dimeric peptides comprising short naturally occurring human and bovine lactoferricin constructs could be further developed as antibiofilm agents.
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http://dx.doi.org/10.1139/bcb-2020-0253DOI Listing
February 2021

Plasma lipid profiling for the prognosis of 90-day mortality, in-hospital mortality, ICU admission, and severity in bacterial community-acquired pneumonia (CAP).

Crit Care 2020 07 27;24(1):461. Epub 2020 Jul 27.

Department of Critical Care Medicine, Faculty of Medicine, Health Research Innovation Center (HRIC), University of Calgary, Room 4C64, 3280 Hospital Drive N.W, Calgary, Alberta, T2N 4Z6, Canada.

Introduction: Pneumonia is the most common cause of mortality from infectious diseases, the second leading cause of nosocomial infection, and the leading cause of mortality among hospitalized adults. To improve clinical management, metabolomics has been increasingly applied to find specific metabolic biopatterns (profiling) for the diagnosis and prognosis of various infectious diseases, including pneumonia.

Methods: One hundred fifty bacterial community-acquired pneumonia (CAP) patients whose plasma samples were drawn within the first 24 h of hospital admission were enrolled in this study and separated into two age- and sex-matched cohorts: non-survivors (died ≤ 90 days) and survivors (survived > 90 days). Three analytical tools, H-NMR spectroscopy, GC-MS, and targeted DI-MS/MS, were used to prognosticate non-survivors from survivors by means of metabolic profiles.

Results: We show that quantitative lipid profiling using DI-MS/MS can predict the 90-day mortality and in-hospital mortality among patients with bacterial CAP compared to H-NMR- and GC-MS-based metabolomics. This study showed that the decreased lysophosphatidylcholines and increased acylcarnitines are significantly associated with increased mortality in bacterial CAP. Additionally, we found that decreased lysophosphatidylcholines and phosphatidylcholines (> 36 carbons) and increased acylcarnitines may be used to predict the prognosis of in-hospital mortality for bacterial CAP as well as the need for ICU admission and severity of bacterial CAP.

Discussion: This study demonstrates that lipid-based plasma metabolites can be used for the prognosis of 90-day mortality among patients with bacterial CAP. Moreover, lipid profiling can be utilized to identify patients with bacterial CAP who are at the highest risk of dying in hospital and who need ICU admission as well as the severity assessment of CAP.
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http://dx.doi.org/10.1186/s13054-020-03147-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385943PMC
July 2020

Antifungal and Antibiofilm Activities and the Mechanism of Action of Repeating Lysine-Tryptophan Peptides against .

Microorganisms 2020 May 18;8(5). Epub 2020 May 18.

Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, Chosun University, Gwangju 61452, Korea.

The rapid increase in the emergence of antifungal-resistant strains is becoming a serious health concern. Because antimicrobial peptides (AMPs) may provide a potential alternative to conventional antifungal agents, we have synthesized a series of peptides with a varying number of lysine and tryptophan repeats (KW-NH). The antifungal activity of these peptides increased with peptide length, but only the longest KW peptide displayed cytotoxicity towards a human keratinocyte cell line. The KW and KW peptides exhibited strong antifungal activity against , even under conditions of high-salt and acidic pH, or the addition of fungal cell wall components. Moreover, KW inhibited biofilm formation by a fluconazole-resistant strain. Circular dichroism and fluorescence spectroscopy indicated that fungal liposomes could interact with the longer peptides but that they did not release the fluorescent dye calcein. Subsequently, fluorescence assays with different dyes revealed that KW did not disrupt the membrane integrity of intact fungal cells. Scanning electron microscopy showed no changes in fungal morphology, while laser-scanning confocal microscopy indicated that KW can localize into the cytosol of . Gel retardation assays revealed that KW can bind to fungal RNA as a potential intracellular target. Taken together, our data indicate that KW can inhibit cellular functions by binding to RNA and DNA after it has been translocated into the cell, resulting in the eradication of .
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http://dx.doi.org/10.3390/microorganisms8050758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285485PMC
May 2020

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Cell 2020 05;181(4):784-799.e19

School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK. Electronic address:

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.
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http://dx.doi.org/10.1016/j.cell.2020.03.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242911PMC
May 2020

Fluorine-19 NMR spectroscopy of fluorinated analogs of tritrpticin highlights a distinct role for Tyr residues in antimicrobial peptides.

Biochim Biophys Acta Biomembr 2020 06 4;1862(6):183260. Epub 2020 Mar 4.

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. Electronic address:

Because of their potential as novel antibiotic agents, antimicrobial peptides (AMPs) have generated considerable interest. The mechanism of bacterial toxicity of AMPs often involves the disruption and/or permeabilization of the bacterial membrane; even those that act intracellularly first have to traverse the membrane. In this work we have explored the incorporation of the fluorinated aromatic amino acids fluoro-Phe and fluoro-Tyr into the Trp- and Arg-rich AMP tritrpticin, and investigated their role in the membrane binding properties and the antimicrobial activity of the peptide. Fluorinated peptides were obtained with good yield by recombinant expression of tritrpticin as a calmodulin-fusion protein in Escherichia coli. Cells were grown in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, and the peptides were released by proteolysis from the purified fusion protein. By using SDS micelles, as a simplified model of the bacterial cytoplasmic membrane, we could study the peptide-membrane interactions and the preferred location of individual fluorinated residues in the micelles by F NMR spectroscopy. Solvent-perturbation F NMR measurements revealed that para-fluoro-Phe residues are embedded deeply in the hydrophobic region of the micelles. On the other hand, 3-fluoro-Tyr residues introduced in tritrpticin were located near the surface of the micelles with high solvent exposure, while 2-fluoro-Tyr sidechains were less solvent exposed. In combination with the outcome of determinations of their antimicrobial activity, our F NMR results indicate that the higher solvent exposure of Tyr residues correlates with a decrease of the antimicrobial potency. This different role of Tyr can likely be extended from tritrpticin to other cationic AMPs.
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http://dx.doi.org/10.1016/j.bbamem.2020.183260DOI Listing
June 2020

Selective anticancer activity of synthetic peptides derived from the host defence peptide tritrpticin.

Biochim Biophys Acta Biomembr 2020 08 29;1862(8):183228. Epub 2020 Feb 29.

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB T2N 1N4, Canada. Electronic address:

Antimicrobial peptides (AMPs) constitute a diverse family of peptides with the ability to protect their host against microbial infections. In addition to their ability to kill microorganisms, several AMPs also exhibit selective cytotoxicity towards cancer cells and are collectively referred to as anticancer peptides (ACPs). Here a large library of AMPs, mainly derived from the porcine cathelicidin peptide, tritrpticin (VRRFPWWWPFLRR), were assessed for their anticancer activity against the Jurkat T cell leukemia line. These anticancer potencies were compared to the cytotoxicity of the peptides towards normal cells isolated from healthy donors, namely peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs; where hemolytic activity was assessed). Among the active tritrpticin derivatives, substitution of Arg by Lys enhanced the selectivity of the peptides towards Jurkat cells when compared to PBMCs. Additionally, the side chain length of the Lys residues was also optimized to further enhance the tritrpticin ACP selectivity at low concentrations. The mechanism of action of the peptides with high selectivity involved the permeabilization of the cytoplasmic membrane of Jurkat cells, without formation of apoptotic bodies. The incorporation of non-natural Lys-based cationic amino acids could provide a new strategy to improve the selectivity of other synthetic ACPs to enhance their potential for therapeutic use against leukemia cells.
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http://dx.doi.org/10.1016/j.bbamem.2020.183228DOI Listing
August 2020

Metabolomic and metallomic profile differences between Veterans and Civilians with Pulmonary Sarcoidosis.

Sci Rep 2019 12 20;9(1):19584. Epub 2019 Dec 20.

Section of Pulmonary, Miami VA Healthcare System, Miami, FL, USA.

Sarcoidosis is a disorder characterized by granulomatous inflammation of unclear etiology. In this study we evaluated whether veterans with sarcoidosis exhibited different plasma metabolomic and metallomic profiles compared with civilians with sarcoidosis. A case control study was performed on veteran and civilian patients with confirmed sarcoidosis. Proton nuclear magnetic resonance spectroscopy (H NMR), hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) and inductively coupled plasma mass spectrometry (ICP-MS) were applied to quantify metabolites and metal elements in plasma samples. Our results revealed that the veterans with sarcoidosis significantly differed from civilians, according to metabolic and metallomics profiles. Moreover, the results showed that veterans with sarcoidosis and veterans with COPD were similar to each other in metabolomics and metallomics profiles. This study suggests the important role of environmental risk factors in the development of different molecular phenotypic responses of sarcoidosis. In addition, this study suggests that sarcoidosis in veterans may be an occupational disease.
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http://dx.doi.org/10.1038/s41598-019-56174-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925242PMC
December 2019

Metabolic Framework for the Improvement of Autism Spectrum Disorders by a Modified Ketogenic Diet: A Pilot Study.

J Proteome Res 2020 01 22;19(1):382-390. Epub 2019 Nov 22.

Shriners Hospitals for Children , Honolulu , Hawaii 96826 , United States.

The ketogenic diet (KD) can improve the core features of autism spectrum disorders (ASD) in some children, but the effects on the overall metabolism remain unclear. This pilot study investigated the behavioral parameters in relation to blood metabolites and trace elements in a cohort of 10 typically developed controls (TC) and 17 children with ASD at baseline and following 3 months of treatment with a modified KD regimen. A nontargeted, multiplatform metabolomic approach was employed, including gas chromatography-mass spectrometry, H nuclear magnetic resonance spectroscopy, and inductively coupled plasma-mass spectrometry. The associations among plasma metabolites, trace elements, and behavior scores were investigated. Employing a combination of metabolomic platforms, 118 named metabolites and 73 trace elements were assessed. Relative to TC, a combination of glutamate, galactonate, and glycerol discriminated ASD with 88% accuracy. ASD had higher concentrations of galactose intermediates, gut microbe-derived trimethylamine -oxide and -acetylserotonin, and lower concentrations of 3-hydroxybutyrate and selenium at baseline. Following 3 months of KD intervention, the levels of circulating ketones and acetylcarnitine were increased. KD restored lower selenium levels in ASD to that of controls, and correlation analysis identified a novel negative correlation between the changes in selenium and behavior scores. Based on the different behavior responses to KD, we found that high responders had greater concentrations of 3-hydroxybutyrate and ornithine, with lower galactose. These findings enhance our current understanding of the metabolic derangements present in ASD and may be of utility in predicting favorable responses to KD intervention.
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http://dx.doi.org/10.1021/acs.jproteome.9b00581DOI Listing
January 2020

Rotamer Jumps, Proton Exchange, and Amine Inversion Dynamics of Dimethylated Lysine Residues in Proteins Resolved by pH-Dependent H and C NMR Relaxation Dispersion.

J Phys Chem B 2019 11 8;123(46):9742-9750. Epub 2019 Nov 8.

Biophysical Chemistry, Center for Molecular Protein Science, Department of Chemistry , Lund University , P.O. Box 124, SE-221 00 Lund , Sweden.

Post-translational methylation of lysine side chains is of great importance for protein regulation, including epigenetic control. Here, we present specific CHD labeling of dimethylated lysines as a sensitive probe of the structure, interactions, and dynamics of these groups, and outline a theoretical and experimental framework for analyzing their conformational dynamics using H and C CPMG relaxation dispersion experiments. Dimethylated lysine side chains in calcium-loaded calmodulin show a marked pH dependence of their Carr-Purcell-Meiboom-Gill (CPMG) dispersion profiles, indicating complex exchange behavior. Combined analysis of H and C CPMG relaxation dispersions requires consideration of 12-state correlated exchange of the two methyl groups due to circular three-state rotamer jumps around the Cε-Nζ axis combined with proton exchange and amine inversion. Taking into account a number of fundamental constraints, the exchange model can be reduced to include only three fitted parameters, namely, the geometric average of the rotamer-jump rate constants, the rate constant of deprotonation of Nζ, and the chemical shift difference between the trans and gauge positions of the C or H nuclei. The pH dependence indicates that protonation of the end group dramatically slows down rotamer exchange for some lysine residues, whereas deprotonation leads to rapid amine inversion coupled with rotamer scrambling. The observed variation among residues in their exchange behavior appears to depend on the structural environment of the side chain. Understanding this type of exchange process is critical to correctly interpreting NMR spectra of methylated lysine side chains. The exchange model presented here forms the basis for studying the structure and dynamics of epigenetically modified lysine side chains and perturbations caused by changes in pH or interactions with target proteins.
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http://dx.doi.org/10.1021/acs.jpcb.9b06408DOI Listing
November 2019

Distinct Gut Microbiota and Serum Metabolites in Response to Weight Loss Induced by Either Dairy or Exercise in a Rodent Model of Obesity.

J Proteome Res 2019 11 27;18(11):3867-3875. Epub 2019 Sep 27.

Alberta Children's Hospital Research Institute , Alberta Children's Hospital , Calgary T3B 6A8 , Alberta , Canada.

Energy imbalance is a primary cause of obesity. While the classical approach to attenuate weight gain includes an increase in energy expenditure through exercise, dietary manipulation such as the inclusion of dairy products has also been proven effective. In the present study, we explored the potential mechanisms by which dairy and exercise attenuate weight gain in diet-induced obese rats. Male Sprague-Dawley rats were fed a high fat, high-sugar (HFHS) diet to induce obesity for 8 weeks. Rats were then further grouped into either control (HFHS + casein) or dairy diet (HFHS + nonfat skim milk) with and without treadmill exercise for 6 weeks. Serum and fresh fecal samples were collected for gut microbiota, serum metabolomics, and metallomics analysis. Diet and exercise resulted in distinct separation in both gut microbiota and serum metabolite profiles. Most intriguingly, obesogenic bacteria including and were reduced, and bioactive molecules such as mannose and arginine were significantly increased in the dairy group. Correlations of at least six bacterial genera with serum metal ions and metabolites were also found. Results reveal distinct impacts of dairy and exercise on the gut microbiota and in the modulation of circulating metabolites with the former primarily responsible for driving microbial alterations known to attenuate weight gain.
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http://dx.doi.org/10.1021/acs.jproteome.9b00304DOI Listing
November 2019

Urinary Metabolomics Validates Metabolic Differentiation Between Renal Cell Carcinoma Stages and Reveals a Unique Metabolic Profile for Oncocytomas.

Metabolites 2019 Jul 24;9(8). Epub 2019 Jul 24.

Department of Biological Sciences, University of Calgary, Calgary, AB T2N 4V8, Canada.

Renal cell carcinoma (RCC) is a heterogeneous malignancy which often develops and progresses asymptomatically. Benign oncocytomas are morphologically similar to malignant chromophobe RCC and distinguishing between these two forms on cross-sectional imaging remains a challenge. Therefore, RCC-specific biomarkers are urgently required for accurate and non-invasive, pre-surgical diagnosis of benign lesions. We have previously shown that dysregulation in glycolytic and tricarboxylic acid cycle intermediates can distinguish benign lesions from RCC in a stage-specific manner. In this study, preoperative fasting urine samples from patients with renal masses were assessed by ¹H nuclear magnetic resonance (NMR). Significant alterations in levels of tricarboxylic acid cycle intermediates, carnitines and its derivatives were detected in RCC relative to benign masses and in oncocytomas vs. chromophobe RCC. Orthogonal Partial Least Square Discriminant Analysis plots confirmed stage discrimination between benign vs. pT1 (R2 = 0.42, Q2 = 0.27) and benign vs. pT3 (R2 = 0.48, Q2 = 0.32) and showed separation for oncocytomas vs. chromophobe RCC (R2 = 0.81, Q2 = 0.57) and oncocytomas vs. clear cell RCC (R2 = 0.32, Q2 = 0.20). This study validates our previously described metabolic profile distinguishing benign tumors from RCC and presents a novel metabolic signature for oncocytomas which may be exploited for diagnosis before cross-sectional imaging.
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http://dx.doi.org/10.3390/metabo9080155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724101PMC
July 2019

A strategy for early detection of response to chemotherapy drugs based on treatment-related changes in the metabolome.

PLoS One 2019 2;14(4):e0213942. Epub 2019 Apr 2.

Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada.

We describe a biomarker-based approach to delivering chemotherapy that entails monitoring treatment changes in the circulating metabolome that reflect efficacy. In-vitro, multiple tumor cell lines were exposed to numerous chemotherapeutics. Supernatants were collected at baseline and 72 hours post treatment. MTT assays were used to quantify growth inhibition. Clinical samples were derived from a phase II clinical trial of second-line axitinib in patients with advanced hepatocellular carcinoma. Sera were collected at baseline and 2-4 weeks after treatment initiation. Response to therapy was estimated by CT scan at 8 weeks. Samples were analyzed by gas chromatography-mass spectrometry to identify metabolomic changes associated with response. In vitro, we found drug-specific and generalizable patterns of change in the extracellular metabolome accompany growth inhibition. A cell death signature was also identified. This approach was also applied to clinical samples. While the in vitro signatures were detectable in vivo, a more robust signal was identified clinically that appeared within 4 weeks of administering drug that distinguished individuals with a treatment response. These changes were extinguished as tumor growth resumed. Serial monitoring of the metabolome during chemotherapy is a means to follow treatment efficacy and emergence of resistance, informing the oncologist whether to modify treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213942PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445409PMC
December 2019

Metabolite Profiling of Clinical Cancer Biofluid Samples by NMR Spectroscopy.

Methods Mol Biol 2019 ;1928:251-274

Department of Biological Sciences, Bio-NMR-Centre, University of Calgary, Calgary, AB, Canada.

Metabolomics is a comprehensive characterization of the small polar molecules (metabolites) in different biological systems. One of the analytical platforms commonly used to study metabolic alterations in biofluid samples is proton nuclear magnetic resonance (H NMR) spectroscopy. NMR spectroscopy is very specific, quantitative, and highly reproducible. Moreover, sample preparation for NMR experiments is very simple and straightforward, and this gives NMR spectroscopy a distinct advantage over other metabolic profiling methods. It has already been shown that H NMR-based profiling of biological fluids can be effective in differentiating benign from malignant lesions and in investigating the efficacy of specific cancer treatments. Therefore, H NMR spectroscopy may become a promising tool for early noninvasive diagnosis and rapid assessment of treatment effects in cancer patients. Here, we describe a detailed protocol for H NMR metabolite profiling in serum, plasma, and urine samples, including sample collection procedures, sample preparation for H NMR experiments, spectral acquisition and processing, and quantitative profiling of H NMR spectra. We also discuss several aspects of appropriate study design and some multivariate statistical methods that are commonly used to analyze metabolomics datasets.
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http://dx.doi.org/10.1007/978-1-4939-9027-6_14DOI Listing
June 2019

Characterization of the EF-Hand Calcium-Binding Domains of Human Plastins.

Methods Mol Biol 2019 ;1929:245-260

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada.

The three human plastins (L-plastin, T-plastin, and I-plastin) are important regulatory Ca-binding proteins that belong to the family of actin-binding proteins. Plastins are involved in the regulation of the actin cytoskeleton as well as the cross-linking of actin filaments. In addition to four calponin-homology (CH) domains, all three plastins contain two N-terminal EF-hand Ca-binding motifs which together are homologous to a single lobe of the well-known calcium-regulatory protein calmodulin. This part of the protein allows for the regulation of the actin bundling activity in response to elevated calcium levels. In this protocol, we describe the purification of the EF-hand headpiece domains of all three plastins, as well as SPR studies, ITC studies, and NMR interaction studies with different peptides and calcium. In combination, these three experimental techniques provide detailed insights into a novel regulatory mechanism, involving the linker region between the EF-hand domain and the first CH domain of the plastins.
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http://dx.doi.org/10.1007/978-1-4939-9030-6_16DOI Listing
June 2019

Metabolic consequences of discretionary fortified beverage consumption containing excessive vitamin B levels in adolescents.

PLoS One 2019 17;14(1):e0209913. Epub 2019 Jan 17.

Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada.

Over the past decade, there has been a substantial increase in the number of beverage products containing added vitamins and minerals. Often viewed as a healthier choice by consumers, the metabolic impacts of excessive vitamin consumption are relatively unknown, especially in children. The aim of this study was to examine the effects of a widely available, vitamin fortified beverage (5h Energy Decaffeinated) on insulin sensitivity, metabolic hormones and serum metabolomic responses in adolescents. Twenty adolescents (13-19y, 10M/10F) completed two randomized trials, consuming either coloured water as placebo (PL) or a vitamin fortified, sugar free beverage (FB, 1.5ml/kg) 40min prior to a modified oral glucose tolerance test (OGTT, 1.75g/kg glucose). Samples were collected at baseline and at 30, 45, 60, 90 and 120min during the OGTT. No differences in blood glucose response were observed between the treatments. However, compared to PL, postprandial plasma C-peptide and insulin excursion was significantly greater with FB, resulting in a 28% decline in the insulin sensitivity index. This was accompanied by elevated GLP-1, glucagon and PYY responses with FB compared to PL. Serum metabolomics (1H-NMR) analysis also revealed perturbations to vitamin B-linked one carbon metabolism flux with FB consumption that became more pronounced over time. These included a transient reduction in homocysteine flux accompanied by increases in betaine, vitamin B6, vitamin B12, choline, folate and taurine. Although these impacts are likely short-lived, results show that beverages fortified with excessive amounts of vitamins are not metabolically inert, but likely result in greater insulin secretion, differential gut hormone secretion and elevated one-carbon flux to process the excessive vitamin loads.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209913PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336241PMC
September 2019

Maternal prebiotic supplementation reduces fatty liver development in offspring through altered microbial and metabolomic profiles in rats.

FASEB J 2019 04 10;33(4):5153-5167. Epub 2019 Jan 10.

Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

A maternal high-fat/sucrose diet, in the presence of maternal obesity, can program increased susceptibility to obesity and metabolic disease in offspring. In particular, nonalcoholic fatty liver disease risk is associated with poor maternal nutrition and obesity status, which may manifest via alterations in gut microbiota. Here, we report that in a preclinical model of diet-induced maternal obesity, maternal supplementation of a high-fat/sucrose diet with the prebiotic oligofructose improves glucose tolerance, insulin sensitivity, and hepatic steatosis in offspring following a long-term high-fat/sucrose dietary challenge compared with offspring of untreated dams. These improvements are associated with alterations in gut microbial composition and serum inflammatory profiles in early life and improvements in inflammatory and fatty-acid gene expression profiles in tissues. Serum metabolomics analysis highlights potential metabolic links between the gut microbiota and the degree of steatosis, including alterations in 1-carbon metabolism. Overall, our data suggest that maternal prebiotic intake protects offspring against hepatic steatosis and insulin resistance following 21 wk of high fat/sucrose diet, which is in part due to alterations in gut microbiota.-Paul, H. A., Collins, K. H., Nicolucci, A. C., Urbanski, S. J., Hart, D. A., Vogel, H. J., Reimer, R. A. Maternal prebiotic supplementation reduces fatty liver development in offspring through altered microbial and metabolomic profiles in rats.
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http://dx.doi.org/10.1096/fj.201801551RDOI Listing
April 2019

Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System.

Microorganisms 2019 Jan 8;7(1). Epub 2019 Jan 8.

Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of biofilms.
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http://dx.doi.org/10.3390/microorganisms7010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6352211PMC
January 2019

Impact of dietary fiber supplementation on modulating microbiota-host-metabolic axes in obesity.

J Nutr Biochem 2019 02 26;64:228-236. Epub 2018 Nov 26.

Faculty of Kinesiology, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada, T2N 1N4; Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada, T2N 4N1. Electronic address:

Low dietary fiber intake is associated with higher rates of microbiota-associated chronic diseases such as obesity. Low-fiber diets alter not only microbial composition but also the availability of metabolic end products derived from fermentation of fiber. Our objective was to examine the effects of dietary fiber supplementation on gut microbiota and associated fecal and serum metabolites in relation to metabolic markers of obesity. We conducted a 12-week, single-center, double-blind, placebo-controlled trial with 53 adults with overweight or obesity. They were randomly assigned to a pea fiber (PF, 15 g/d in wafer form; n=29) or control (CO, isocaloric amount of wafers; n=24) group. Blood and fecal samples were collected at baseline and 12 weeks. Serum metabolomics, gut microbiota and fecal short-chain fatty acids (SCFAs) and bile acids (BAs) were examined. Within-group but not between-group analysis showed a significant effect of treatment on serum metabolites at 12 weeks compared to baseline. Fiber significantly altered fecal SCFAs and BAs with higher acetate and reduced isovalerate, cholate, deoxycholate and total BAs content in the PF group compared to baseline. Microbiota was differentially modulated in the two groups, including an increase in the SCFA producer Lachnospira in the PF group and decrease in the CO group. The change in body weight of participants showed a negative correlation with their change in Lachnospira (r=-0.463, P=.006) abundance. The current study provides insight into the actions of pea fiber and its impact on modulating microbiota-host-metabolic axes in obesity.
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http://dx.doi.org/10.1016/j.jnutbio.2018.11.003DOI Listing
February 2019

Biomarker Phenotype for Early Diagnosis and Triage of Sepsis to the Pediatric Intensive Care Unit.

Sci Rep 2018 11 9;8(1):16606. Epub 2018 Nov 9.

Division of Pediatric Critical Care Medicine, Department of Pediatrics, University of Alberta, Edmonton, AB, Canada.

Early diagnosis and triage of sepsis improves outcomes. We aimed to identify biomarkers that may advance diagnosis and triage of pediatric sepsis. Serum and plasma samples were collected from young children (1-23 months old) with sepsis on presentation to the Pediatric Intensive Care Unit (PICU-sepsis, n = 46) or Pediatric Emergency Department (PED-sepsis, n = 58) and PED-non-sepsis patients (n = 19). Multivariate analysis was applied to distinguish between patient groups. Results were compared to our results for older children (2-17 years old). Common metabolites and protein-mediators were validated as potential biomarkers for a sepsis-triage model to differentiate PICU-sepsis from PED-sepsis in children age 1 month-17 years. Metabolomics in young children clearly separated the PICU-sepsis and PED-sepsis cohorts: sensitivity 0.71, specificity 0.93, and AUROC = 0.90 ± 0.03. Adding protein-mediators to the model did not improve performance. The seven metabolites common to the young and older children were used to create the sepsis-triage model. Validation of the sepsis-triage model resulted in sensitivity: 0.83 ± 0.02, specificity: 0.88 ± 0.05 and AUROC 0.93 ± 0.02. The metabolic-based biomarkers predicted which sepsis patients required care in a PICU versus those that could be safely cared for outside of a PICU. This has potential to inform appropriate triage of pediatric sepsis, particularly in EDs with less experience evaluating children.
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http://dx.doi.org/10.1038/s41598-018-35000-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226431PMC
November 2018

Solution structures of the SH3 domains from Shank scaffold proteins and their interactions with Cav1.3 calcium channels.

FEBS Lett 2018 08 12;592(16):2786-2797. Epub 2018 Aug 12.

Department of Biological Sciences, University of Calgary, Canada.

Shank proteins are abundant scaffold proteins in the postsynaptic density (PSD) region of brain synapses. Mutations in Shank proteins are associated with autism, schizophrenia, and Alzheimer's disease. To gain insights into Shank protein interactions at the PSD, we determined the solution structures of the src homology 3 (SH3) domains of all three mammalian Shank proteins. Our findings indicate that they have identical and typical SH3 folding motifs, but unusual target-binding pockets. An investigation into the interaction between the Shank SH3 domains and the proline-rich region of the Cav1.3 calcium channel revealed an atypical interaction in which the highly acidic specificity binding pocket of the SH3 domains binds to a Cav1.3 region containing a cluster of three Arg residues. Our study provides insights into Shank SH3-mediated interactions.
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http://dx.doi.org/10.1002/1873-3468.13209DOI Listing
August 2018
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