Publications by authors named "Elan Eisenmesser"

56 Publications

The Inherent Dynamics and Interaction Sites of the SARS-CoV-2 Nucleocapsid N-Terminal Region.

J Mol Biol 2021 07 20;433(15):167108. Epub 2021 Jun 20.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, School of Medicine, Aurora, CO 80045, United States. Electronic address:

The nucleocapsid protein is one of four structural proteins encoded by SARS-CoV-2 and plays a central role in packaging viral RNA and manipulating the host cell machinery, yet its dynamic behavior and promiscuity in nucleotide binding has made standard structural methods to address its atomic-resolution details difficult. To begin addressing the SARS-CoV-2 nucleocapsid protein interactions with both RNA and the host cell along with its dynamic behavior, we have specifically focused on the folded N-terminal domain (NTD) and its flanking regions using nuclear magnetic resonance solution studies. Studies performed here reveal a large repertoire of interactions, which includes a temperature-dependent self-association mediated by the disordered flanking regions that also serve as binding sites for host cell cyclophilin-A while nucleotide binding is largely mediated by the central NTD core. NMR studies that include relaxation experiments have revealed the complicated dynamic nature of this viral protein. Specifically, while much of the N-terminal core domain exhibits micro-millisecond motions, a central β-hairpin shows elevated inherent flexibility on the pico-nanosecond timescale and the serine/arginine-rich region of residues 176-209 undergoes multiple exchange phenomena. Collectively, these studies have begun to reveal the complexities of the nucleocapsid protein dynamics and its preferred interaction sites with its biological targets.
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http://dx.doi.org/10.1016/j.jmb.2021.167108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214912PMC
July 2021

Modulating Enzyme Function Dynamic Allostery within Biliverdin Reductase B.

Front Mol Biosci 2021 20;8:691208. Epub 2021 May 20.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Denver, CO, United States.

The biliverdin reductase B (BLVRB) class of enzymes catalyze the NADPH-dependent reduction of multiple flavin substrates and are emerging as critical players in cellular redox regulation. However, the role of dynamics and allostery have not been addressed, prompting studies here that have revealed a position 15 Å away from the active site within human BLVRB (T164) that is inherently dynamic and can be mutated to control global micro-millisecond motions and function. By comparing the inherent dynamics through nuclear magnetic resonance (NMR) relaxation approaches of evolutionarily distinct BLVRB homologues and by applying our previously developed Relaxation And Single Site Multiple Mutations (RASSMM) approach that monitors both the functional and dynamic effects of multiple mutations to the single T164 site, we have discovered that the most dramatic mutagenic effects coincide with evolutionary changes and these modulate coenzyme binding. Thus, evolutionarily changing sites distal to the active site serve as dynamic "dials" to globally modulate motions and function. Despite the distal dynamic and functional coupling modulated by this site, micro-millisecond motions span an order of magnitude in their apparent kinetic rates of motions. Thus, global dynamics within BLVRB are a collection of partially coupled motions tied to catalytic function.
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http://dx.doi.org/10.3389/fmolb.2021.691208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173106PMC
May 2021

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Haematologica 2021 Nov 1;106(11):2971-2985. Epub 2021 Nov 1.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.
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http://dx.doi.org/10.3324/haematol.2020.278252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8561282PMC
November 2021

Quantifying dynamic range in red blood cell energetics: Evidence of progressive energy failure during storage.

Transfusion 2021 05 8;61(5):1586-1599. Epub 2021 Apr 8.

Department of Pediatrics, Divisions of Critical Care Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Background: During storage, red blood cells (RBCs) undergo significant biochemical and morphologic changes, referred to collectively as the "storage lesion". It was hypothesized that these defects may arise from disrupted oxygen-based regulation of RBC energy metabolism, with resultant depowering of intrinsic antioxidant systems.

Study Design And Methods: As a function of storage duration, the dynamic range in RBC metabolic response to three models of biochemical oxidant stress (methylene blue, hypoxanthine/xanthine oxidase, and diamide) was assessed, comparing glycolytic flux by NMR and UHPLC-MS methodologies. Blood was processed/stored under standard conditions (AS-1 additive solution) with leukoreduction. Over a 6-week period, RBC metabolic and antioxidant status were assessed at baseline and following exposure to the three biochemical oxidant models. Comparison was made of glycolytic flux ( H-NMR tracking of [2- C]-glucose and metabolomic phenotyping with [1,2,3- C ] glucose), reducing equivalent (NADPH/NADP ) recycling, and thiol-based (GSH/GSSG) antioxidant status.

Results: As a function of storage duration, we observed the following: (1) a reduction in baseline hexose monophosphate pathway (HMP) flux, the sole pathway responsible for the regeneration of the essential reducing equivalent NADPH; with (2) diminished stress-based dynamic range in both overall glycolytic as well as proportional HMP flux. In addition, progressive with storage duration, RBCs showed (3) constraint in reducing equivalent (NADPH) recycling capacity, (4) loss of thiol based (GSH) recycling capacity, and (5) dysregulation of metabolon assembly at the cytoplasmic domain of Band 3 membrane protein (cdB3).

Conclusion: Blood storage disturbs normal RBC metabolic control, depowering antioxidant capacity and enhancing vulnerability to oxidative injury.
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http://dx.doi.org/10.1111/trf.16395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363157PMC
May 2021

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling.

J Biol Chem 2021 Jan-Jun;296:100630. Epub 2021 Apr 3.

Department of Medicine, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, USA. Electronic address:

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.
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http://dx.doi.org/10.1016/j.jbc.2021.100630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018910PMC
July 2021

Development and Validation of a Multiplex Microsphere Immunoassay Using Dried Blood Spots for SARS-CoV-2 Seroprevalence: Application in First Responders in Colorado, USA.

J Clin Microbiol 2021 05 19;59(6). Epub 2021 May 19.

Department of Immunology and Microbiology, University of Colorado, Anschutz Medical Campus, Aurora, Colorado, USA.

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies ( = 0.83,  < 0.0001 and  = 0.87,  < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS ( = 0.76,  = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.
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http://dx.doi.org/10.1128/JCM.00290-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8315929PMC
May 2021

Interleukin-37 improves T-cell-mediated immunity and chimeric antigen receptor T-cell therapy in aged backgrounds.

Aging Cell 2021 02 22;20(2):e13309. Epub 2021 Jan 22.

Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.

Aging-associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world's population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging-associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin-37 (IL-37) is a potent anti-inflammatory cytokine, and we present data demonstrating that IL-37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin-37 (IL-37) in aged mice reduces or prevents aging-associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL-37 expression decreases the surface expression of programmed cell death protein 1 (PD-1) and augments cytokine production from aged T-cells. Improved T-cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4 T-cells and Lat in CD8 T-cells when aged mice were treated with recombinant IL-37 (rIL-37) but not control immunoglobin (Control Ig). Importantly, IL-37-mediated rejuvenation of aged endogenous T-cells was also observed in aged chimeric antigen receptor (CAR) T-cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL-37 in boosting the function of aged T-cells and highlight its therapeutic potential to overcome aging-associated immunosenescence.
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http://dx.doi.org/10.1111/acel.13309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884049PMC
February 2021

IL-37 exerts therapeutic effects in experimental autoimmune encephalomyelitis through the receptor complex IL-1R5/IL-1R8.

Theranostics 2021 1;11(1):1-13. Epub 2021 Jan 1.

Institut de Neurociencies and Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, Bellaterra, Catalonia, Spain.

Interleukin 37 (IL-37), a member of IL-1 family, broadly suppresses inflammation in many pathological conditions by acting as a dual-function cytokine in that IL-37 signals via the extracellular receptor complex IL1-R5/IL-1R8, but it can also translocate to the nucleus. However, whether IL-37 exerts beneficial actions in neuroinflammatory diseases, such as multiple sclerosis, remains to be elucidated. Thus, the goals of the present study were to evaluate the therapeutic effects of IL-37 in a mouse model of multiple sclerosis, and if so, whether this is mediated via the extracellular receptor complex IL-1R5/IL-1R8. We used a murine model of MS, the experimental autoimmune encephalomyelitis (EAE). We induced EAE in three different single and double transgenic mice (hIL-37tg, IL-1R8 KO, hIL-37tg-IL-1R8 KO) and wild type littermates. We also induced EAE in C57Bl/6 mice and treated them with various forms of recombinant human IL-37 protein. Functional and histological techniques were used to assess locomotor deficits and demyelination. Luminex and flow cytometry analysis were done to assess the protein levels of pro-inflammatory cytokines and different immune cell populations, respectively. qPCRs were done to assess the expression of IL-37, IL-1R5 and IL-1R8 in the spinal cord of EAE, and in blood peripheral mononuclear cells and brain tissue samples of MS patients. We demonstrate that IL-37 reduces inflammation and protects against neurological deficits and myelin loss in EAE mice by acting via IL1-R5/IL1-R8. We also reveal that administration of recombinant human IL-37 exerts therapeutic actions in EAE mice. We finally show that IL-37 transcripts are not up-regulated in peripheral blood mononuclear cells and in brain lesions of MS patients, despite the IL-1R5/IL-1R8 receptor complex is expressed. This study presents novel data indicating that IL-37 exerts therapeutic effects in EAE by acting through the extracellular receptor complex IL-1R5/IL-1R8, and that this protective physiological mechanism is defective in MS individuals. IL-37 may therefore represent a novel therapeutic avenue for the treatment of MS with great promising potential.
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http://dx.doi.org/10.7150/thno.47435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681099PMC
August 2021

Blood donor exposome and impact of common drugs on red blood cell metabolism.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado, USA.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.
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http://dx.doi.org/10.1172/jci.insight.146175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934844PMC
February 2021

Mechanism and inhibition of Streptococcus pneumoniae IgA1 protease.

Nat Commun 2020 11 27;11(1):6063. Epub 2020 Nov 27.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, School of Medicine, Aurora, CO, 80045, USA.

Opportunistic pathogens such as Streptococcus pneumoniae secrete a giant metalloprotease virulence factor responsible for cleaving host IgA1, yet the molecular mechanism has remained unknown since their discovery nearly 30 years ago despite the potential for developing vaccines that target these enzymes to block infection. Here we show through a series of cryo-electron microscopy single particle reconstructions how the Streptococcus pneumoniae IgA1 protease facilitates IgA1 substrate recognition and how this can be inhibited. Specifically, the Streptococcus pneumoniae IgA1 protease subscribes to an active-site-gated mechanism where a domain undergoes a 10.0 Å movement to facilitate cleavage. Monoclonal antibody binding inhibits this conformational change, providing a direct means to block infection at the host interface. These structural studies explain decades of biological and biochemical studies and provides a general strategy to block Streptococcus pneumoniae IgA1 protease activity to potentially prevent infection.
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http://dx.doi.org/10.1038/s41467-020-19887-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695701PMC
November 2020

Human recombinant interleukin-38 suppresses inflammation in mouse models of local and systemic disease.

Cytokine 2021 01 28;137:155334. Epub 2020 Oct 28.

Department of Medicine, University of Colorado Denver, Aurora, CO, USA; Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Interleukin (IL)-38 belongs to the IL-1 family and is part of the IL-36 subfamily due to its binding to the IL-36 Receptor (IL-1R6). In the current study, we assessed the anti-inflammatory properties of IL-38 in murine models of arthritis and systemic inflammation. First, the anti-inflammatory properties of mouse and human IL-38 precursors were compared to forms with a truncated N-terminus. In mouse bone marrow derived dendritic cells (BMDC), human and mouse IL-38 precursors with a truncation of the two N-terminal amino acids (3-152) suppressed LPS-induced IL-6. Recombinant human IL-38 (3-152) was further investigated for its immunomodulatory potential using four murine models of inflammatory disease: streptococcal cell wall (SCW)-induced arthritis, monosodium urate (MSU) crystal-induced arthritis, MSU crystal-induced peritonitis, and systemic endotoxemia. In each of these models IL-38 significantly reduced inflammation. In SCW and MSU crystal-induced arthritis, joint swelling, inflammatory cell influx, and synovial levels of IL-1β, IL-6, and KC were reduced by 50% or greater. These suppressive properties of IL-38 in SCW-induced arthritis were independent of the anti-inflammatory co-receptor IL-1R8, as IL-38 reduced arthritis equally in IL-1R8 deficient and WT mice. In MSU crystal-induced peritonitis, IL-38 reduced hypothermia, while plasma IL-6 and KC and peritoneal KC levels were reduced by 65-70%. In the LPS endotoxemia model, IL-38 pretreatment reduced systemic IL-6, TNFα and KC. Furthermore, in ex vivo cultured bone marrow, LPS-induced IL-6, TNFα and KC were reduced by 75-90%. Overall, IL-38 exhibits broad anti-inflammatory properties in models of systemic and local inflammation and therefore may be an effective cytokine therapy.
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http://dx.doi.org/10.1016/j.cyto.2020.155334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725974PMC
January 2021

Extracellular and nuclear roles of IL-37 after spinal cord injury.

Brain Behav Immun 2021 01 28;91:194-201. Epub 2020 Sep 28.

Departament de Biologia Cel·lular, Fisiologia i Immunologia, Institut de Neurociències, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Universitat Autonoma de Barcelona, Bellaterra, Catalonia 08193, Spain. Electronic address:

Interleukin 37 (IL-37) is an anti-inflammatory cytokine of the interleukin 1 family. Transgenic mice expressing the human form of the IL37 gene (hIL-37Tg) display protective effects in several animal models of disease. Previous data from our group revealed that IL-37 limits inflammation after spinal cord injury (SCI) and ameliorates tissue damage and functional deficits. IL-37 can exert its anti-inflammatory effects by translocating to the nucleus or acting as an extracellular cytokine. However, whether this protection after SCI is mediated by translocating to the nucleus, activating of extracellular receptors, or both, is currently unknown. In the present study, we used different transgenic animals to answer this question. We demonstrated that the beneficial effects of IL-37 on functional and histological outcomes after SCI were lost in the lack of the extracellular receptor IL-1R8, indicating that IL-37 induces protection as an extracellular cytokine. On the other hand, transgenic mice with the nuclear function of IL-37 abolished (hIL-37D20ATg) showed significant improvement in locomotor skills and myelin sparing after SCI, indicating that nuclear pathway is not required for the protective actions of IL-37. Moreover, we also showed that the therapeutic effects of the recombinant IL-37 protein are produced only in the presence of the extracellular receptor IL-1R8, further highlighting the importance of the extracellular function of this cytokine after SCI. Finally, we revealed that the administration of recombinant IL-37 protein exerted therapeutic actions when administered in the lesion site but not systemically. This work demonstrated for the first time that translocation of IL-37 to the nucleus is not required for the beneficial actions of this cytokine after SCI and highlights the importance of the extracellular signaling of IL-37 to mediate neuroprotective actions.
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http://dx.doi.org/10.1016/j.bbi.2020.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749842PMC
January 2021

Activation Loop Dynamics Are Coupled to Core Motions in Extracellular Signal-Regulated Kinase-2.

Biochemistry 2020 07 15;59(29):2698-2706. Epub 2020 Jul 15.

Department of Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, United States.

The activation loop segment in protein kinases is a common site for regulatory phosphorylation. In extracellular signal-regulated kinase 2 (ERK2), dual phosphorylation and conformational rearrangement of the activation loop accompany enzyme activation. X-ray structures show the active conformation to be stabilized by multiple ion pair interactions between phosphorylated threonine and tyrosine residues in the loop and six arginine residues in the kinase core. Despite the extensive salt bridge network, nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiments show that the phosphorylated activation loop is conformationally mobile on a microsecond to millisecond time scale. The dynamics of the loop match those of previously reported global exchange within the kinase core region and surrounding the catalytic site that have been found to facilitate productive nucleotide binding. Mutations in the core region that alter these global motions also alter the dynamics of the activation loop. Conversely, mutations in the activation loop perturb the global exchange within the kinase core. Together, these findings provide evidence for coupling between motions in the activation loop and those surrounding the catalytic site in the active state of the kinase. Thus, the activation loop segment in dual-phosphorylated ERK2 is not held statically in the active X-ray conformation but instead undergoes exchange between conformers separated by a small energetic barrier, serving as part of a dynamic allosteric network controlling nucleotide binding and catalytic function.
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http://dx.doi.org/10.1021/acs.biochem.0c00485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867030PMC
July 2020

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family.

J Biochem 2020 Aug;168(2):191-202

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, 12801 E 17th Ave., Aurora, CO 80045, USA.

Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme 'clamps' formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.
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http://dx.doi.org/10.1093/jb/mvaa039DOI Listing
August 2020

Rare genetic variants in interleukin-37 link this anti-inflammatory cytokine to the pathogenesis and treatment of gout.

Ann Rheum Dis 2020 04 29;79(4):536-544. Epub 2020 Feb 29.

Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands

Objective: Gout is characterised by severe interleukin (IL)-1-mediated joint inflammation induced by monosodium urate crystals. Since IL-37 is a pivotal anti-inflammatory cytokine suppressing the activity of IL-1, we conducted genetic and functional studies aimed at elucidating the role of IL-37 in the pathogenesis and treatment of gout.

Methods: Variant identification was performed by DNA sequencing of all coding bases of using molecular inversion probe-based resequencing (discovery cohort: gout n=675, controls n=520) and TaqMan genotyping (validation cohort: gout n=2202, controls n=2295). Predictive modelling of the effects of rare variants on protein structure was followed by in vitro experiments evaluating the impact on protein function. Treatment with recombinant IL-37 was evaluated in vitro and in vivo in a mouse model of gout.

Results: We identified four rare variants in in six of the discovery gout patients; p.(A144P), p.(G174Dfs*16), p.(C181*) and p.(N182S), whereas none emerged in healthy controls (Fisher's exact p-value=0.043). All variants clustered in the functional domain of IL-37 in exon 5 (p-value=5.71×10). Predictive modelling and functional studies confirmed loss of anti-inflammatory functions and we substantiated the therapeutic potential of recombinant IL-37 in the treatment of gouty inflammation. Furthermore, the carrier status of p.(N182S)(rs752113534) was associated with increased risk (OR=1.81, p-value=0.031) of developing gout in hyperuricaemic individuals of Polynesian ancestry.

Conclusion: Here, we provide genetic as well as mechanistic evidence for the role of IL-37 in the pathogenesis of gout, and highlight the therapeutic potential of recombinant IL-37 for the treatment of gouty arthritis.
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http://dx.doi.org/10.1136/annrheumdis-2019-216233DOI Listing
April 2020

Short-term interleukin-37 treatment improves vascular endothelial function, endurance exercise capacity, and whole-body glucose metabolism in old mice.

Aging Cell 2020 01 21;19(1):e13074. Epub 2019 Nov 21.

Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, USA.

Aging is associated with vascular endothelial dysfunction, reduced exercise tolerance, and impaired whole-body glucose metabolism. Interleukin-37 (IL-37), an anti-inflammatory cytokine of the interleukin-1 family, exerts salutary physiological effects in young mice independent of its inflammation-suppressing properties. Here, we assess the efficacy of IL-37 treatment for improving physiological function in older age. Old mice (26-28 months) received daily intraperitoneal injections of recombinant human IL-37 (recIL-37; 1 µg/200 ml PBS) or vehicle (200 ml PBS) for 10-14 days. Vascular endothelial function (ex vivo carotid artery dilation to increasing doses of acetylcholine, ACh) was enhanced in recIL-37 vs. vehicle-treated mice via increased nitric oxide (NO) bioavailability (all p < .05); this effect was accompanied by enhanced ACh-stimulated NO production and reduced levels of reactive oxygen species in endothelial cells cultured with plasma from IL-37-treated animals (p < .05 vs. vehicle plasma). RecIL-37 treatment increased endurance exercise capacity by 2.4-fold, which was accompanied by a 2.9-fold increase in the phosphorylated AMP-activated kinase (AMPK) to AMPK ratio (i.e., AMPK activation) in quadriceps muscle. RecIL-37 treatment also improved whole-body insulin sensitivity and glucose tolerance (p < .05 vs. vehicle). Improvements in physiological function occurred without significant changes in plasma, aortic, and skeletal muscle pro-inflammatory proteins (under resting conditions), whereas pro-/anti-inflammatory IL-6 was greater in recIL-37-treated animals. Plasma metabolomics analysis revealed that recIL-37 treatment altered metabolites related to pathways involved in NO synthesis (e.g., increased L-arginine and citrulline/arginine ratio) and fatty acid metabolism (e.g., increased pantothenol and free fatty acids). Our findings provide experimental support for IL-37 therapy as a novel strategy to improve diverse physiological functions in old age.
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http://dx.doi.org/10.1111/acel.13074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974720PMC
January 2020

Streptococcus pneumoniae G5 domains bind different ligands.

Protein Sci 2019 10 9;28(10):1797-1805. Epub 2019 Aug 9.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, School of Medicine, Aurora, Colorado.

Many bacterial pathogens express small G5 domains that exist in the context of various membrane-anchored proteins and these G5 domains have been associated with colonization, cellular adhesion, and biofilm formation. However, despite over a decade since the computational prediction of these G5 domains, many remain uncharacterized, particularly those from Streptococcus pneumoniae. Of five previously predicted G5 domains we found that four of these, all derived from S. pneumoniae, are independently folded modules. As one of these exhibits extreme line broadening due to self-association, we were able to use NMR solution studies to probe the potential ligand interactions of the remaining three G5 domains. None of these G5 domains engage N-acetylglucosamine (NAG) as previously predicted but do interact with other small molecules that may modulate adherence to both bacteria and host cells. Specifically, while all G5 domains tested engage Zn, only one of these G5 domains engage heparin. NMR solution structural studies of the IgA1 Protease G5 (IgA1P-G5) and endo-beta-N-acetylglucosaminidase-D G5 (ENDD-G5) also facilitated identification of the ligand binding sites and confirm the typical G5 fold that comprises two connected β-sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the β-sheets. Our studies thus establish a basis for future biological experiments to test whether the ligands presented here are involved in bacterial adherence, either to bacteria or to host cells.
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http://dx.doi.org/10.1002/pro.3693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6740092PMC
October 2019

Protect, repair, destroy or sacrifice: a role of oxidative stress biology in inter-donor variability of blood storage?

Blood Transfus 2019 07 6;17(4):281-288. Epub 2019 Jun 6.

BloodWorks Northwest, Seattle, WA, United States of America.

Red blood cells (RBCs) have been historically regarded as a critical model to investigate cellular and oxidant stress biology. First of all, they are constantly exposed to oxidant stress, as their main function is to transport and deliver oxygen to tissues. Second, they are devoid of de novo protein synthesis capacity, which prevents RBCs from replacing irreversibly oxidised proteins with newly synthesised ones. As such, RBCs have evolved to (i) protect themselves from oxidant stress, in order to prevent oxidant damage from reactive species; (ii) repair oxidatively damaged proteins, through mechanisms that involve glutathione and one-carbon metabolism; (iii) destroy irreversibly oxidised proteins through proteasomal or protease-dependent degradation; and (iv) sacrifice membrane portions through mechanism of vesiculation. In this brief review we will summarize these processes and their relevance to RBC redox biology (within the context of blood storage), with a focus on how polymorphisms in RBC antioxidant responses could contribute to explaining the heterogeneity in the progression and severity of the RBC storage lesion that can be observed across the healthy donor population.
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http://dx.doi.org/10.2450/2019.0072-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683869PMC
July 2019

Interleukin-37 monomer is the active form for reducing innate immunity.

Proc Natl Acad Sci U S A 2019 03 28;116(12):5514-5522. Epub 2019 Feb 28.

Department of Medicine, University of Colorado Denver, Aurora, CO 80238;

Interleukin-37 (IL-37), a member of the IL-1 family of cytokines, is a fundamental suppressor of innate and acquired immunities. Here, we used an integrative approach that combines biophysical, biochemical, and biological studies to elucidate the unique characteristics of IL-37. Our studies reveal that single amino acid mutations at the IL-37 dimer interface that result in the stable formation of IL-37 monomers also remain monomeric at high micromolar concentrations and that these monomeric IL-37 forms comprise higher antiinflammatory activities than native IL-37 on multiple cell types. We find that, because native IL-37 forms dimers with nanomolar affinity, higher IL-37 only weakly suppresses downstream markers of inflammation whereas lower concentrations are more effective. We further show that IL-37 is a heparin binding protein that modulates this self-association and that the IL-37 dimers must block the activity of the IL-37 monomer. Specifically, native IL-37 at 2.5 nM reduces lipopolysaccharide (LPS)-induced vascular cell adhesion molecule (VCAM) protein levels by ∼50%, whereas the monomeric D73K mutant reduced VCAM by 90% at the same concentration. Compared with other members of the IL-1 family, both the N and the C termini of IL-37 are extended, and we show they are disordered in the context of the free protein. Furthermore, the presence of, at least, one of these extended termini is required for IL-37 suppressive activity. Based on these structural and biological studies, we present a model of IL-37 interactions that accounts for its mechanism in suppressing innate inflammation.
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http://dx.doi.org/10.1073/pnas.1819672116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431183PMC
March 2019

IL-38 has an anti-inflammatory action in psoriasis and its expression correlates with disease severity and therapeutic response to anti-IL-17A treatment.

Cell Death Dis 2018 10 30;9(11):1104. Epub 2018 Oct 30.

Laboratory of Experimental Immunology and Integrated Research Center for PSOriasis (CRI-PSO), Istituto Dermopatico dell'Immacolata IDI-IRCCS, via Monti di Creta, 104, ROME, Italy.

IL-36 cytokines, a subgroup of IL-1 family, comprise IL-36α, IL-36β, and IL-36γ agonists, abundantly expressed in psoriatic skin, and IL-36RA and IL-38 antagonists. In psoriatic skin, IL-36 cytokines interfere with keratinocyte cornification programs and induce the release of antimicrobial peptides and chemokines active on neutrophils and Th17 lymphocytes. To date, the role of IL-38 antagonist in psoriasis remains to be defined. Here, we demonstrate that skin and circulating IL-38 levels are reduced in psoriatic patients and in other skin diseases characterized by neutrophilic infiltrate. In psoriasis, the balance of IL-36γ agonist/IL-38 antagonist serum levels is in favor of agonists and is closely associated with disease severity. Interestingly, IL-38 is upregulated by anti-IL-17A biological treatment and positively correlates with the therapeutic efficacy of secukinumab in psoriatic patients. The downregulation of IL-38 expression is strictly related to keratinocyte de-differentiation triggered by the inflammatory cytokines IL-36γ, IL-17, and IL-22. Finally, we demonstrate that administration of recombinant full-length IL-38 counteracts in vitro the biological processes induced by IL-36γ in human keratinocytes and endothelial cells and attenuates in vivo the severity of the psoriasiform phenotype induced by IMQ in mice. Such effects are achieved by restoring the physiological programs of keratinocyte proliferation and differentiation, and reducing the immune cell infiltrates.
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http://dx.doi.org/10.1038/s41419-018-1143-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207563PMC
October 2018

Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells.

Transfusion 2018 12 12;58(12):2978-2991. Epub 2018 Oct 12.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.

Background: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank.

Study Design And Methods: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway.

Results: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3.

Conclusion: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.
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http://dx.doi.org/10.1111/trf.14936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357231PMC
December 2018

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

J Mol Biol 2018 09 20;430(18 Pt B):3234-3250. Epub 2018 Jun 20.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USA. Electronic address:

Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78➔Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.
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http://dx.doi.org/10.1016/j.jmb.2018.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431292PMC
September 2018

The Mechanism of HdeA Unfolding and Chaperone Activation.

J Mol Biol 2018 01 11;430(1):33-40. Epub 2017 Nov 11.

Department of Chemistry & Biochemistry and the Knoebel Institute for Healthy Aging, University of Denver, Denver, CO 80208, USA. Electronic address:

HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition from folded dimer to unfolded monomer between pH3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.
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http://dx.doi.org/10.1016/j.jmb.2017.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738273PMC
January 2018

The spliceosomal proteins PPIH and PRPF4 exhibit bi-partite binding.

Biochem J 2017 10 25;474(21):3689-3704. Epub 2017 Oct 25.

Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, U.S.A.

Pre-mRNA splicing is a dynamic, multistep process that is catalyzed by the RNA (ribonucleic acid)-protein complex called the spliceosome. The spliceosome contains a core set of RNAs and proteins that are conserved in all organisms that perform splicing. In higher organisms, peptidyl-prolyl isomerase H (PPIH) directly interacts with the core protein pre-mRNA processing factor 4 (PRPF4) and both integrate into the pre-catalytic spliceosome as part of the tri-snRNP (small nuclear RNA-protein complex) subcomplex. As a first step to understand the protein interactions that dictate PPIH and PRPF4 function, we expressed and purified soluble forms of each protein and formed a complex between them. We found two sites of interaction between PPIH and the N-terminus of PRPF4, an unexpected result. The N-terminus of PRPF4 is an intrinsically disordered region and does not adopt secondary structure in the presence of PPIH. In the absence of an atomic resolution structure, we used mutational analysis to identify point mutations that uncouple these two binding sites and find that mutations in both sites are necessary to break up the complex. A discussion of how this bipartite interaction between PPIH and PRPF4 may modulate spliceosomal function is included.
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http://dx.doi.org/10.1042/BCJ20170366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682932PMC
October 2017

Interleukin 37 reverses the metabolic cost of inflammation, increases oxidative respiration, and improves exercise tolerance.

Proc Natl Acad Sci U S A 2017 02 13;114(9):2313-2318. Epub 2017 Feb 13.

Department of Medicine, University of Colorado Denver, Aurora, CO 80045;

IL-1 family member interleukin 37 (IL-37) has broad antiinflammatory properties and functions as a natural suppressor of innate inflammation. In this study, we demonstrate that treatment with recombinant human IL-37 reverses the decrease in exercise performance observed during systemic inflammation. This effect was associated with a decrease in the levels of plasma and muscle cytokines, comparable in extent to that obtained upon IL-1 receptor blockade. Exogenous administration of IL-37 to healthy mice, not subjected to an inflammatory challenge, also improved exercise performance by 82% compared with vehicle-treated mice ( = 0.01). Treatment with eight daily doses of IL-37 resulted in a further 326% increase in endurance running time compared with the performance level of mice receiving vehicle ( 0.001). These properties required the engagement of the IL-1 decoy receptor 8 (IL-1R8) and the activation of AMP-activated protein kinase (AMPK), because both inhibition of AMPK and IL-1R8 deficiency abrogated the positive effects of IL-37 on exercise performance. Mechanistically, treatment with IL-37 induced marked metabolic changes with higher levels of muscle AMPK, greater rates of oxygen consumption, and increased oxidative phosphorylation. Metabolomic analyses of plasma and muscles of mice treated with IL-37 revealed an increase in AMP/ATP ratio, reduced levels of proinflammatory mediator succinate and oxidative stress-related metabolites, as well as changes in amino acid and purine metabolism. These effects of IL-37 to limit the metabolic costs of chronic inflammation and to foster exercise tolerance provide a rationale for therapeutic use of IL-37 in the treatment of inflammation-mediated fatigue.
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http://dx.doi.org/10.1073/pnas.1619011114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338542PMC
February 2017

Networks of Dynamic Allostery Regulate Enzyme Function.

Structure 2017 02 12;25(2):276-286. Epub 2017 Jan 12.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver, 12801 East 17th Avenue, MS 8101, Aurora, CO 80045, USA. Electronic address:

Many protein systems rely on coupled dynamic networks to allosterically regulate function. However, the broad conformational space sampled by non-coherently dynamic systems has precluded detailed analysis of their communication mechanisms. Here, we have developed a methodology that combines the high sensitivity afforded by nuclear magnetic resonance relaxation techniques and single-site multiple mutations, termed RASSMM, to identify two allosterically coupled dynamic networks within the non-coherently dynamic enzyme cyclophilin A. Using this methodology, we discovered two key hotspot residues, Val6 and Val29, that communicate through these networks, the mutation of which altered active-site dynamics, modulating enzymatic turnover of multiple substrates. Finally, we utilized molecular dynamics simulations to identify the mechanism by which one of these hotspots is coupled to the larger dynamic networks. These studies confirm a link between enzyme dynamics and the catalytic cycle of cyclophilin A and demonstrate how dynamic allostery may be engineered to tune enzyme function.
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http://dx.doi.org/10.1016/j.str.2016.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336394PMC
February 2017

CD147: a small molecule transporter ancillary protein at the crossroad of multiple hallmarks of cancer and metabolic reprogramming.

Oncotarget 2017 Jan;8(4):6742-6762

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, CO, USA.

Increased expression of CD147 in pancreatic cancer has been proposed to play a critical role in cancer progression via CD147 chaperone function for lactate monocarboxylate transporters (MCTs). Here, we show for the first time that CD147 interacts with membrane transporters beyond MCTs and exhibits a protective role for several of its interacting partners. CD147 prevents its interacting partner's proteasome-dependent degradation and incorrect plasma membrane localization through the CD147 transmembrane (TM) region. The interactions with transmembrane small molecule and ion transporters identified here indicate a central role of CD147 in pancreatic cancer metabolic reprogramming, particularly with respect to amino acid anabolism and calcium signaling. Importantly, CD147 genetic ablation prevents pancreatic cancer cell proliferation and tumor growth in vitro and in vivo in conjunction with metabolic rewiring towards amino acid anabolism, thus paving the way for future combined pharmacological treatments.
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http://dx.doi.org/10.18632/oncotarget.14272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341751PMC
January 2017

Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro.

Protein Sci 2017 03 23;26(3):600-610. Epub 2017 Feb 23.

Departments of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO.

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn-binding motif (1604-1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N-terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C-terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.
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http://dx.doi.org/10.1002/pro.3110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326571PMC
March 2017

Dynamical network of residue-residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation.

Proc Natl Acad Sci U S A 2016 Apr 11;113(17):4735-40. Epub 2016 Apr 11.

Department of Chemistry, Georgia State University, Atlanta, GA 30302;

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.
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http://dx.doi.org/10.1073/pnas.1523573113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855540PMC
April 2016

Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems.

Biochemistry 2015 Sep 11;54(38):5815-27. Epub 2015 Sep 11.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver , 12801 East 17th Avenue, Aurora, Colorado 80045, United States.

Cyclophilins catalyze cis ↔ trans isomerization of peptidyl-prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.
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http://dx.doi.org/10.1021/acs.biochem.5b00746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801485PMC
September 2015
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