ABSTRACT
Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.
Subject(s)
Haemophilus Infections , Haemophilus influenzae , Haemophilus , Humans , Heme/metabolism , Lung/microbiology , IronABSTRACT
The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1ß which contribute to organoid growth. However, high concentrations of IL-1ß have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.
Subject(s)
Intestinal Mucosa , Wnt Signaling Pathway , Animals , Mice , Homeostasis , Intercellular Signaling Peptides and Proteins/metabolism , Interleukins/metabolism , Intestinal Mucosa/metabolism , Organoids/metabolism , Stem Cells/metabolismABSTRACT
Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.
Subject(s)
Acinetobacter baumannii , NAD , Acinetobacter baumannii/genetics , Acinetobacter baumannii/metabolism , Bacteria/metabolism , Bacterial Proteins/metabolism , Deuterium , Hydrolases/metabolism , Mammals/metabolism , NAD/metabolism , Protein DomainsABSTRACT
Biliverdin Reductase B (BLVRB) is an NADPH-dependent reductase that catalyzes the reduction of multiple substrates and is therefore considered a critical cellular redox regulator. In this study, we sought to address whether both structural and dynamics changes occur between different intermediates of the catalytic cycle and whether these were relegated to just the active site or the entirety of the enzyme. Through X-ray crystallography, we determined the apo BLVRB structure for the first time, revealing subtle global changes compared to the holo structure and identifying the loss of a critical hydrogen bond that "clamps" the R78-loop over the coenzyme. Amide and Cα chemical shift perturbations were used to identify environmental and secondary structural changes between intermediates, with more distant global changes observed upon coenzyme binding compared to substrate interactions. NMR relaxation rate measurements provided insights into the dynamic behavior of BLVRB during the catalytic cycle. Specifically, the inherently dynamic R78-loop that becomes ordered upon coenzyme binding persists through the catalytic cycle while similar regions experience dynamic exchange. However, the dynamic exchange processes were found to differ through the catalytic cycle with several groups of residues exhibiting similar dynamic responses. Finally, both local and distal structural and dynamic changes occur within BLVRB that are dependent solely on the oxidative state of the coenzyme. Thus, through a comprehensive analysis here, this study revealed structural and dynamic alterations in BLVRB through its catalytic cycle that are not simply relegated to the active site, but instead, are allosterically coupled throughout the enzyme.
ABSTRACT
Advances in Nuclear Magnetic Resonance (NMR) spectroscopy have allowed for the identification and characterization of movements in enzymes over the last 20 years that has also revealed the complexities of allosteric coupling. For example, many of the inherent movements of enzymes, and proteins in general, have been shown to be highly localized but nonetheless still coupled over long distances. Such partial couplings provide challenges to both identifying allosteric networks of dynamic communication and determining their roles in catalytic function. We have developed an approach to help identify and engineer enzyme function, called Relaxation And Single Site Multiple Mutations (RASSMM). This approach is a powerful extension of mutagenesis and NMR that is based on the observation that multiple mutations to a single site distal to the active site allosterically induces different effects to networks. Such an approach generates a panel of mutations that can also be subjected to functional studies in order to match catalytic effects with changes to coupled networks. In this review, the RASSMM approach is briefly outlined together with two applications that include cyclophilin-A and Biliverdin Reductase B.
Subject(s)
Proteins , Mutation , Proteins/chemistry , Mutagenesis , Magnetic Resonance Spectroscopy , Catalytic Domain , Allosteric RegulationABSTRACT
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid protein is the most abundantly expressed viral protein during infection where it targets both RNA and host proteins. However, identifying how a single viral protein interacts with so many different targets remains a challenge, providing the impetus here for identifying the interaction sites through multiple methods. Through a combination of nuclear magnetic resonance (NMR), electron microscopy, and biochemical methods, we have characterized nucleocapsid interactions with RNA and with three host proteins, which include human cyclophilin-A, Pin1, and 14-3-3τ. Regarding RNA interactions, the nucleocapsid protein N-terminal folded domain preferentially interacts with smaller RNA fragments relative to the C-terminal region, suggesting an initial RNA engagement is largely dictated by this N-terminal region followed by weaker interactions to the C-terminal region. The nucleocapsid protein forms 10 nm ribonuclear complexes with larger RNA fragments that include 200 and 354 nucleic acids, revealing its potential diversity in sequestering different viral genomic regions during viral packaging. Regarding host protein interactions, while the nucleocapsid targets all three host proteins through its serine-arginine-rich region, unstructured termini of the nucleocapsid protein also engage host cyclophilin-A and host 14-3-3τ. Considering these host proteins play roles in innate immunity, the SARS-CoV-2 nucleocapsid protein may block the host response by competing interactions. Finally, phosphorylation of the nucleocapsid protein quenches an inherent dynamic exchange process within its serine-arginine-rich region. Our studies identify many of the diverse interactions that may be important for SARS-CoV-2 pathology during infection.
Subject(s)
COVID-19 , RNA , Humans , SARS-CoV-2/metabolism , Cyclophilins/analysis , Nucleocapsid/chemistry , Nucleocapsid/metabolism , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Arginine , Serine , NIMA-Interacting Peptidylprolyl Isomerase/analysisABSTRACT
The mucosal adaptive immune response is dependent on the production of IgA antibodies and particularly IgA1, yet opportunistic bacteria have evolved mechanisms to specifically block this response by producing IgA1 proteases (IgA1Ps). Our lab was the first to describe the structures of a metal-dependent IgA1P (metallo-IgA1P) produced from Gram-positive Streptococcus pneumoniae both in the absence and presence of its IgA1 substrate through cryo-EM single particle reconstructions. This prior study revealed an active-site gating mechanism reliant on substrate-induced conformational changes to the enzyme that begged the question of whether such a mechanism is conserved among the wider Gram-positive metallo-IgA1P subfamily of virulence factors. Here, we used cryo-EM to characterize the metallo-IgA1P of a more distantly related family member from Gemella haemolysans, an emerging opportunistic pathogen implicated in meningitis, endocarditis, and more recently bacteremia in the elderly. While the substrate-free structures of these two metallo-IgA1Ps exhibit differences in the relative starting positions of the domain responsible for gating substrate, the enzymes have similar domain orientations when bound to IgA1. Together with biochemical studies that indicate these metallo-IgA1Ps have similar binding affinities and activities, these data indicate that metallo-IgA1P binding requires the specific IgA1 substrate to open the enzymes for access to their active site and thus, largely conform to an "induced fit" model.
Subject(s)
Immunoglobulin A , Metalloproteases , Humans , Aged , Immunoglobulin A/metabolism , Streptococcus/metabolism , Bacteria/metabolism , Virulence FactorsABSTRACT
Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.
Subject(s)
Bacteria , Virulence Factors , Animals , Bacteria/metabolism , Bacterial Proteins/metabolism , Gram-Negative Bacteria/metabolism , Humans , Mammals/metabolism , Signal Transduction , Virulence Factors/chemistryABSTRACT
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 ß-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.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Mutation , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , Evolution, Molecular , HEK293 Cells , Humans , Immune Evasion , Models, Molecular , Protein Conformation , Protein Domains , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
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.
ABSTRACT
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-1ß, 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.
Subject(s)
Arthritis, Gouty/prevention & control , Arthritis/prevention & control , Disease Models, Animal , Inflammation/prevention & control , Interleukins/pharmacology , Recombinant Proteins/pharmacology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/pharmacology , Arthritis/metabolism , Arthritis, Gouty/metabolism , Cells, Cultured , Cytokines/blood , Humans , Inflammation/chemically induced , Inflammation/metabolism , Interleukins/genetics , Lipopolysaccharides , Male , Mice, Inbred C57BL , Peritonitis/metabolism , Peritonitis/prevention & control , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Blood Donors , Erythrocytes/drug effects , Erythrocytes/metabolism , Exposome , Nonprescription Drugs/adverse effects , Nonprescription Drugs/pharmacokinetics , Prescription Drugs/adverse effects , Prescription Drugs/pharmacokinetics , Adolescent , Adult , Aged , Animals , Energy Metabolism/drug effects , Erythrocyte Transfusion , Female , Glycolysis/drug effects , Healthy Volunteers , Hemoglobins/metabolism , High-Throughput Screening Assays , Humans , In Vitro Techniques , Machine Learning , Male , Metabolomics , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Models, Biological , Oxidation-Reduction/drug effects , Phosphotransferases (Alcohol Group Acceptor)/deficiency , Phosphotransferases (Alcohol Group Acceptor)/genetics , Ranitidine/pharmacology , Young AdultABSTRACT
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.
Subject(s)
Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Streptococcus pneumoniae/enzymology , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/ultrastructure , Biocatalysis , Catalytic Domain , Cryoelectron Microscopy , Models, Molecular , Protein Binding , Serine Endopeptidases/chemistry , Serine Endopeptidases/ultrastructureABSTRACT
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.
Subject(s)
Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Thermodynamics , Crystallography, X-Ray , Humans , Models, Molecular , Oxidoreductases Acting on CH-CH Group Donors/isolation & purification , Protein ConformationABSTRACT
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 ß-sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the ß-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.
Subject(s)
Bacterial Proteins/chemistry , Streptococcus pneumoniae/chemistry , Bacterial Adhesion , Bacterial Proteins/metabolism , Ligands , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Streptococcus pneumoniae/metabolismABSTRACT
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 â¼50%, 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.
Subject(s)
Immune Tolerance , Immunity, Innate , Interleukin-1/metabolism , Cell Line , Crystallography, X-Ray , Humans , Immune Tolerance/immunology , Immune Tolerance/physiology , Interleukin-1/genetics , Interleukin-1/physiology , Magnetic Resonance Spectroscopy , Protein MultimerizationABSTRACT
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 Arg78âAla 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.
Subject(s)
Coenzymes/chemistry , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Binding Sites , Catalytic Domain , Coenzymes/genetics , Coenzymes/metabolism , Flavin-Adenine Dinucleotide/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Mutation , NADP/chemistry , NADP/metabolism , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Protein Conformation , Structure-Activity RelationshipABSTRACT
Brain tumors such as high grade gliomas are among the deadliest forms of human cancers. The tumor environment is subject to a number of cellular stressors such as hypoxia and glucose deprivation. The persistence of the stressors activates the unfolded proteins response (UPR) and results in global alterations in transcriptional and translational activity of the cell. Although the UPR is known to effect tumorigenesis in some epithelial cancers, relatively little is known about the role of the UPR in brain tumors. Here, we evaluated the changes at the molecular level under homeostatic and stress conditions in two glioma cell lines of differing tumor grade. Using mass spectrometry analysis, we identified proteins unique to each condition (unstressed/stressed) and within each cell line (U87MG and UPN933). Comparing the two, we find differences between both the conditions and cell lines indicating a unique profile for each. Finally, we used our proteomic data to identify the predominant pathways within these cells under unstressed and stressed conditions. Numerous predominant pathways are the same in both cell lines, but there are differences in biological and molecular classifications of the identified proteins, including signaling mechanisms, following UPR induction; we see that relatively minimal proteomic alterations can lead to signaling changes that ultimately promote cell survival.
Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , Neoplasm Proteins/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Glioma/genetics , Glioma/pathology , Humans , Mass Spectrometry , Proteomics/methods , Unfolded Protein ResponseABSTRACT
In Huntington's disease (HD) the imperfect expanded CAG repeat in the first exon of the HTT gene leads to the generation of a polyglutamine (polyQ) protein, which has some neuronal toxicity, potentially mollified by formation of aggregates. Accumulated research, reviewed here, implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in extracellular vesicles (EVs). Most cell types in the brain release EVs and these have been shown to contain neurodegenerative proteins in the case of prion protein and amyloid-beta peptide. In this study, we used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of Hdh mRNA containing the expanded repeat incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another, but further work will be needed to evaluate potential EV and cell-type specificity of transfer and effects of long-term exposure. It seems likely that expanded HD-associated repeat RNA may appear in biofluids and may have use as biomarkers of disease state and response to therapy.