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1.
Arch Biochem Biophys ; 758: 110087, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38977154

ABSTRACT

Protein aggregation in the form of amyloid fibrils has long been associated with the onset and development of various amyloidoses, including Alzheimer's, Parkinson's or prion diseases. Recent studies of their fibril formation process have revealed that amyloidogenic protein cross-interactions may impact aggregation pathways and kinetic parameters, as well as the structure of the resulting aggregates. Despite a growing number of reports exploring this type of interaction, they only cover just a small number of possible amyloidogenic protein pairings. One such pair is between two neurodegeneration-associated proteins: the pro-inflammatory S100A9 and prion protein, which are known to co-localize in vivo. In this study, we examined their cross-interaction in vitro and discovered that the fibrillar form of S100A9 modulated the aggregation pathway of mouse prion protein 89-230 fragment, while non-aggregated S100A9 also significantly inhibited its primary nucleation process. These results complement previous observations of the pro-inflammatory protein's role in amyloid aggregation and highlight its potential role against neurodegenerative disorders.


Subject(s)
Amyloid , Calgranulin B , Prion Proteins , Protein Aggregates , Calgranulin B/metabolism , Calgranulin B/chemistry , Animals , Mice , Prion Proteins/chemistry , Prion Proteins/metabolism , Amyloid/metabolism , Amyloid/chemistry , Peptide Fragments/metabolism , Peptide Fragments/chemistry , Kinetics
2.
Protein J ; 43(2): 243-258, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38431537

ABSTRACT

S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.


Subject(s)
CD36 Antigens , Calgranulin A , Calgranulin B , Molecular Docking Simulation , Receptor for Advanced Glycation End Products , Toll-Like Receptor 4 , Calgranulin B/chemistry , Calgranulin B/metabolism , Toll-Like Receptor 4/chemistry , Toll-Like Receptor 4/metabolism , Calgranulin A/chemistry , Calgranulin A/metabolism , Calgranulin A/genetics , Humans , CD36 Antigens/chemistry , CD36 Antigens/metabolism , CD36 Antigens/genetics , Receptor for Advanced Glycation End Products/chemistry , Receptor for Advanced Glycation End Products/metabolism , Protein Binding , Molecular Dynamics Simulation , Surface Plasmon Resonance , Protein Multimerization , Arthritis, Rheumatoid/metabolism
3.
J Biol Chem ; 299(4): 102952, 2023 04.
Article in English | MEDLINE | ID: mdl-36731796

ABSTRACT

S100A8 and S100A9 are small, human, Ca2+-binding proteins with multiple intracellular and extracellular functions in signaling, regulation, and defense. The two proteins are not detected as monomers but form various noncovalent homo- or hetero-oligomers related to specific activities in human physiology. Because of their significant roles in numerous medical conditions, there has been intense research on the conformational properties of various S100A8 and S100A9 proteoforms as essential targets of drug discovery. NMR or crystal structures are currently available only for mutated or truncated protein complexes, mainly with bound metal ions, that may well reflect the proteins' properties outside cells but not in other biological contexts in which they perform. Here, we used structural mass spectrometry methods combined with molecular dynamics simulations to compare the conformations of wildtype full-length S100A8 and S100A9 subunits in biologically relevant homo- and heterodimers and in higher oligomers formed in the presence of calcium or zinc ions. We provide, first, rationales for their functional response to changing environmental conditions, by elucidating differences between proteoforms in flexible protein regions that may provide the plasticity of the binding sites for the multiple targets, and second, the key factors contributing to the variable stability of the oligomers. The described methods and a systematic view of the conformational properties of S100A8 and S100A9 complexes provide a basis for further research to characterize and modulate their functions for basic science and therapies.


Subject(s)
Calgranulin A , Calgranulin B , Humans , Binding Sites , Calgranulin A/chemistry , Calgranulin B/chemistry , Protein Conformation , Molecular Dynamics Simulation , Mass Spectrometry
4.
Adv Sci (Weinh) ; 8(21): e2101796, 2021 11.
Article in English | MEDLINE | ID: mdl-34519180

ABSTRACT

Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose-limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium-binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche, as well as, post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9-targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next-generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease.


Subject(s)
Breast Neoplasms/pathology , Calgranulin B/metabolism , Comovirus/immunology , Melanoma, Experimental/pathology , Nanoparticles/chemistry , Animals , Breast Neoplasms/mortality , Calgranulin B/chemistry , Cell Line, Tumor , Comovirus/chemistry , Female , Humans , Immunotherapy , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Melanoma, Experimental/mortality , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nanoparticles/metabolism , Nanoparticles/therapeutic use , Peptides/chemistry , Pre-Exposure Prophylaxis , Survival Rate
5.
Int J Mol Sci ; 22(15)2021 Jul 26.
Article in English | MEDLINE | ID: mdl-34360737

ABSTRACT

The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson's disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution's ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils.


Subject(s)
Amyloid/chemistry , Calgranulin B/chemistry , Protein Aggregates , alpha-Synuclein/chemistry , Humans , Recombinant Proteins/chemistry
6.
Int J Mol Sci ; 22(16)2021 Aug 09.
Article in English | MEDLINE | ID: mdl-34445262

ABSTRACT

The amyloid cascade is central for the neurodegeneration disease pathology, including Alzheimer's and Parkinson's, and remains the focus of much current research. S100A9 protein drives the amyloid-neuroinflammatory cascade in these diseases. DOPA and cyclen-based compounds were used as amyloid modifiers and inhibitors previously, and DOPA is also used as a precursor of dopamine in Parkinson's treatment. Here, by using fluorescence titration experiments we showed that five selected ligands: DOPA-D-H-DOPA, DOPA-H-H-DOPA, DOPA-D-H, DOPA-cyclen, and H-E-cyclen, bind to S100A9 with apparent Kd in the sub-micromolar range. Ligand docking and molecular dynamic simulation showed that all compounds bind to S100A9 in more than one binding site and with different ligand mobility and H-bonds involved in each site, which all together is consistent with the apparent binding determined in fluorescence experiments. By using amyloid kinetic analysis, monitored by thioflavin-T fluorescence, and AFM imaging, we found that S100A9 co-aggregation with these compounds does not hinder amyloid formation but leads to morphological changes in the amyloid fibrils, manifested in fibril thickening. Thicker fibrils were not observed upon fibrillation of S100A9 alone and may influence the amyloid tissue propagation and modulate S100A9 amyloid assembly as part of the amyloid-neuroinflammatory cascade in neurodegenerative diseases.


Subject(s)
Amyloid/chemistry , Calgranulin B/chemistry , Dihydroxyphenylalanine/chemistry , Molecular Dynamics Simulation , Protein Aggregates , Humans
7.
Sci Rep ; 11(1): 11472, 2021 06 01.
Article in English | MEDLINE | ID: mdl-34075153

ABSTRACT

In post-stroke patients, a decreased adherence to antiplatelet drugs is a major challenge in the prevention of recurrent stroke. Previously, we reported an antiplatelet vaccine against S100A9 in mice, but the use of Freund's adjuvant and the difference in amino acid sequences in epitopes between mice and humans were problematic for clinical use. Here, we redesigned the S100A9 vaccine for the common sequence in both humans and monkeys and examined its effects in cynomolgus monkeys with Alum adjuvant. First, we assessed several candidate epitopes and selected 102 to 112 amino acids as the suitable epitope, which could produce antibodies. When this peptide vaccine was intradermally injected into 4 cynomolgus monkeys with Alum, the antibody against human S100A9 was successfully produced. Anti-thrombotic effects were shown in two monkeys in a mixture of vaccinated serum and fresh whole blood from another cynomolgus monkey. Additionally, the anti-thrombotic effects were partially inhibited by the epitope peptide, indicating the feasibility of neutralizing anti-thrombotic effects of produced antibodies. Prolongation of bleeding time was not observed in vaccinated monkeys. Although further studies on increasing the effect of vaccine and safety are necessary, this vaccine will be a promising approach to improve adherence to antiplatelet drugs in clinical settings.


Subject(s)
Calgranulin B , Fibrinolytic Agents , Peptides , Thrombosis , Vaccines , Animals , Calgranulin B/chemistry , Calgranulin B/immunology , Calgranulin B/pharmacology , Fibrinolytic Agents/immunology , Fibrinolytic Agents/pharmacology , Humans , Macaca fascicularis , Macaca mulatta , Peptides/chemistry , Peptides/immunology , Peptides/pharmacology , Thrombosis/immunology , Thrombosis/therapy , Vaccines/immunology , Vaccines/pharmacology
8.
ACS Appl Mater Interfaces ; 13(23): 26721-26734, 2021 Jun 16.
Article in English | MEDLINE | ID: mdl-34080430

ABSTRACT

Pro-inflammatory and amyloidogenic S100A9 protein is central to the amyloid-neuroinflammatory cascade in neurodegenerative diseases. Polyoxometalates (POMs) constitute a diverse group of nanomaterials, which showed potency in amyloid inhibition. Here, we have demonstrated that two selected nanosized niobium POMs, Nb10 and TiNb9, can act as potent inhibitors of S100A9 amyloid assembly. Kinetics analysis based on ThT fluorescence experiments showed that addition of either Nb10 or TiNb9 reduces the S100A9 amyloid formation rate and amyloid quantity. Atomic force microscopy imaging demonstrated the complete absence of long S100A9 amyloid fibrils at increasing concentrations of either POM and the presence of only round-shaped and slightly elongated aggregates. Molecular dynamics simulation revealed that both Nb10 and TiNb9 bind to native S100A9 homo-dimer by forming ionic interactions with the positively charged Lys residue-rich patches on the protein surface. The acrylamide quenching of intrinsic fluorescence showed that POM binding does not perturb the Trp 88 environment. The far and near UV circular dichroism revealed no large-scale perturbation of S100A9 secondary and tertiary structures upon POM binding. These indicate that POM binding involves only local conformational changes in the binding sites. By using intrinsic and 8-anilino-1-naphthalene sulfonate fluorescence titration experiments, we found that POMs bind to S100A9 with a Kd of ca. 2.5 µM. We suggest that the region, including Lys 50 to Lys 54 and characterized by high amyloid propensity, could be the key sequences involved in S1009 amyloid self-assembly. The inhibition and complete hindering of S100A9 amyloid pathways may be used in the therapeutic applications targeting the amyloid-neuroinflammatory cascade in neurodegenerative diseases.


Subject(s)
Amyloid/antagonists & inhibitors , Calgranulin B/chemistry , Calgranulin B/metabolism , Neurodegenerative Diseases , Tungsten Compounds/pharmacology , Humans , Protein Conformation
9.
Molecules ; 26(5)2021 Mar 02.
Article in English | MEDLINE | ID: mdl-33801279

ABSTRACT

Deregulations of the expression of the S100A8 and S100A9 genes and/or proteins, as well as changes in their plasma levels or their levels of secretion in the bone marrow microenvironment, are frequently observed in acute myeloblastic leukemias (AML) and acute lymphoblastic leukemias (ALL). These deregulations impact the prognosis of patients through various mechanisms of cellular or extracellular regulation of the viability of leukemic cells. In particular, S100A8 and S100A9 in monomeric, homodimeric, or heterodimeric forms are able to modulate the survival and the sensitivity to chemotherapy of leukemic clones through their action on the regulation of intracellular calcium, on oxidative stress, on the activation of apoptosis, and thanks to their implications, on cell death regulation by autophagy and pyroptosis. Moreover, biologic effects of S100A8/9 via both TLR4 and RAGE on hematopoietic stem cells contribute to the selection and expansion of leukemic clones by excretion of proinflammatory cytokines and/or immune regulation. Hence, the therapeutic targeting of S100A8 and S100A9 appears to be a promising way to improve treatment efficiency in acute leukemias.


Subject(s)
Antineoplastic Agents/pharmacology , Calgranulin A/metabolism , Calgranulin B/metabolism , Leukemia, Myeloid, Acute/pathology , Molecular Targeted Therapy , Animals , Calgranulin A/antagonists & inhibitors , Calgranulin B/chemistry , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/metabolism , Signal Transduction
10.
ACS Chem Biol ; 14(7): 1410-1417, 2019 07 19.
Article in English | MEDLINE | ID: mdl-31194501

ABSTRACT

Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from Aß peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self-assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.


Subject(s)
Amyloid/immunology , CD56 Antigen/immunology , Calgranulin B/immunology , Protein Aggregation, Pathological/immunology , Amino Acid Sequence , Amyloid/chemistry , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/immunology , CD56 Antigen/chemistry , Calgranulin B/chemistry , Humans , Inflammation/immunology , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/immunology , Prions/chemistry , Prions/immunology , Protein Aggregates
11.
J Mater Chem B ; 7(11): 1842-1846, 2019 03 21.
Article in English | MEDLINE | ID: mdl-32255046

ABSTRACT

We integrate a biocompatible plant virus-based nanotechnology (tobacco mosaic virus, TMV) with S100A9-targeting peptides for its application in imaging and diagnosis of atherosclerosis. S100A9-targeted TMV nanoparticles exhibit remarkable specificity to S100A9 and targeting of atherosclerosis lesions in ApoE-/- mice.


Subject(s)
Atherosclerosis/diagnostic imaging , Calgranulin B , Nanoparticles , Optical Imaging/methods , Tobacco Mosaic Virus/metabolism , Animals , Calgranulin B/chemistry , Calgranulin B/metabolism , Mice , Mice, Knockout, ApoE , Nanoparticles/chemistry , Nanoparticles/metabolism
12.
J Am Chem Soc ; 140(50): 17444-17455, 2018 12 19.
Article in English | MEDLINE | ID: mdl-30380834

ABSTRACT

Oxidative post-translational modifications affect the structure and function of many biomolecules. Herein we examine the biophysical and functional consequences of oxidative post-translational modifications to human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer, calgranulins A/B oligomer). This abundant metal-sequestering protein contributes to innate immunity by starving invading microbial pathogens of transition metal nutrients in the extracellular space. It also participates in the inflammatory response. Despite many decades of study, little is known about the fate of CP at sites of infection and inflammation. We present compelling evidence for methionine oxidation of CP in vivo, supported by using 15N-labeled CP-Ser (S100A8(C42S)/S100A9(C3S)) to monitor for adventitious oxidation following human sample collection. To elucidate the biochemical and functional consequences of oxidative post-translational modifications, we examine recombinant CP-Ser with methionine sulfoxide modifications generated by exposing the protein to hydrogen peroxide. These oxidized species coordinate transition metal ions and exert antibacterial activity. Nevertheless, oxidation of M81 in the S100A9 subunit disrupts Ca(II)-induced tetramerization and, in the absence of a transition metal ion bound at the His6 site, accelerates proteolytic degradation of CP. We demonstrate that native CP, which contains one Cys residue in each full-length subunit, forms disulfide bonds within and between S100A8/S100A9 heterodimers when exposed to hydrogen peroxide. Remarkably, disulfide bond formation accelerates proteolytic degradation of CP. We propose a new extension to the working model for extracellular CP where post-translational oxidation by reactive oxygen species generated during the neutrophil oxidative burst modulates its lifetime in the extracellular space.


Subject(s)
Calgranulin A/metabolism , Calgranulin B/metabolism , Protein Processing, Post-Translational , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Calcium/metabolism , Calgranulin A/chemistry , Calgranulin B/chemistry , Cysteine/chemistry , Disulfides/chemistry , Escherichia coli/drug effects , Humans , Methionine/chemistry , Mucus/chemistry , Oxidation-Reduction , Protein Multimerization , Proteolysis , Staphylococcus aureus/drug effects , Suppuration
13.
Biochemistry ; 57(28): 4155-4164, 2018 07 17.
Article in English | MEDLINE | ID: mdl-29890074

ABSTRACT

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca(II) ions on the structure and Ni(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni(II)-bound CP-Ser [oligomer of S100A8(C42S) and S100A9(C3S)] crystals obtained in the absence and presence of Ca(II). We present a 2.1 Å resolution crystal structure of Ni(II)-bound CP-Ser and compare this structure to a reported Ni(II)- and Ca(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) J. Am. Chem. Soc. 139, 8828-8836]. This analysis reveals conformational changes associated with coordination of Ca(II) to the EF-hands of S100A9 and that Ca(II) binding affects the coordination number and geometry of the Ni(II) ion bound to the His3Asp site. In contrast, negligible differences are observed for the Ni(II)-His6 site in the absence and presence of Ca(II). Biochemical studies show that, whereas the His6 site has a thermodynamic preference for Ni(II) over Zn(II), the His3Asp site selects for Zn(II) over Ni(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space.


Subject(s)
Calcium/metabolism , Calgranulin A/metabolism , Calgranulin B/metabolism , Nickel/metabolism , Binding Sites , Calcium/chemistry , Calgranulin A/chemistry , Calgranulin B/chemistry , Crystallography, X-Ray , EF Hand Motifs , Humans , Models, Molecular , Nickel/chemistry , Protein Binding , Protein Conformation
14.
J Clin Invest ; 128(5): 1852-1866, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29611822

ABSTRACT

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.


Subject(s)
Alarmins/immunology , Calgranulin A/immunology , Calgranulin B/immunology , Alarmins/chemistry , Alarmins/genetics , Animals , Arthritis/genetics , Arthritis/immunology , Arthritis/pathology , Binding Sites , Calgranulin A/chemistry , Calgranulin A/genetics , Calgranulin B/chemistry , Calgranulin B/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Mice , Mice, Knockout , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/immunology , Psoriasis/genetics , Psoriasis/immunology , Psoriasis/pathology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology
15.
Biochemistry ; 57(19): 2846-2856, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29659256

ABSTRACT

Calprotectin (CP) is an abundant host-defense protein that contributes to the metal-withholding innate immune response by sequestering nutrient metal ions from microbial pathogens in the extracellular space. Over the past decade, murine models of infectious disease have advanced understanding of the physiological functions of CP and its ability to compete with microbes for essential metal nutrients. Despite this extensive work, murine CP (mCP) has not been biochemically evaluated, and structural and biophysical understanding of CP is currently limited to the human orthologue. We present the reconstitution, purification, and characterization of mCP as well as the cysteine-null variant mCP-Ser. Apo mCP is a mS100A8/mS100A9 heterodimer, and Ca(II) binding causes two heterodimers to self-associate and form a heterotetramer. Initial metal-depletion studies demonstrate that mCP depletes multiple first-row transition metal ions, including Mn, Fe, Ni, Cu, and Zn, from complex microbial growth medium, indicating that mCP binds multiple nutrient metals with high affinity. Moreover, antibacterial activity assays show that mCP inhibits the growth of a variety of bacterial species. The metal-depletion and antibacterial activity studies also provide evidence that Ca(II) ions enhance these functional properties of mCP. This contribution provides the groundwork for understanding the similarities and differences between the human and murine orthologues of CP and for further elucidation of its biological coordination chemistry.


Subject(s)
Calcium/chemistry , Calgranulin A/chemistry , Calgranulin B/chemistry , Leukocyte L1 Antigen Complex/chemistry , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/pathogenicity , Humans , Ions/chemistry , Iron/chemistry , Leukocyte L1 Antigen Complex/pharmacology , Manganese/chemistry , Mice , Models, Molecular , Protein Multimerization , Zinc/chemistry
16.
Mol Inform ; 37(6-7): e1700144, 2018 07.
Article in English | MEDLINE | ID: mdl-29418080

ABSTRACT

Many biological phenomena can be represented as complex networks. Using a protein binding site comparison approach, we generated a network of ion binding sites on the scale of all known protein structures from the Protein Data Bank. We found that this ion binding site similarity network is scale-free, indicating a network in which a few ion binding site scaffolds are the network hubs, and these are connected to hundreds of nodes, whereas the vast majority of nodes have only a few neighbors. Enrichment and statistical analysis of the network components and communities yielded insights into underlying processes from the functional and the structural perspective. Largest components and communities were observed to be closely related to basic metabolic processes and some of the most common structural folds, which, from the evolutionary point of view, indicates that they may be the oldest ones. Further, we derived the first comprehensive map of ion interchangeability, based on binding site similarity. Several highly interchangeable protein-ion binding site pairs emerged (e.g., Ca2+ and Mg2+ ), as well as structurally distinct ones. The constructed network of ion binding site similarities will aid in understanding the general principles of protein-ion binding sites structure, function and evolution. We demonstrate potential uses of the network on proteins involved in cancer development and immune response, where individual ions play prominent roles in disease development.


Subject(s)
Ions/pharmacology , Molecular Docking Simulation/methods , Proteome/chemistry , Sequence Analysis, Protein/methods , Animals , Binding Sites , Calgranulin B/chemistry , Calgranulin B/genetics , Calgranulin B/metabolism , Evolution, Molecular , Humans , Protein Binding , Proteome/genetics , Proteome/metabolism
17.
Nat Med ; 24(2): 154-164, 2018 02.
Article in English | MEDLINE | ID: mdl-29291352

ABSTRACT

Hemophagocytic syndrome (HPS) is a fatal hyperinflammatory disease with a poorly understood mechanism that occurs most frequently in extranodal natural killer/T cell lymphoma (ENKTL). Through exome sequencing of ENKTL tumor-normal samples, we have identified a hotspot mutation (c.419T>C) in the evolutionarily conserved signaling intermediate in Toll pathway (ECSIT) gene, encoding a V140A variant of ECSIT. ECSIT-V140A activated NF-κB more potently than the wild-type protein owing to its increased affinity for the S100A8 and S100A9 heterodimer, which promotes NADPH oxidase activity. ECSIT-T419C knock-in mice showed higher peritoneal NADPH oxidase activity than mice with wild-type ECSIT in response to LPS. ECSIT-T419C-transfected ENKTL cell lines produced tumor necrosis factor (TNF)-α and interferon (IFN)-γ, which induced macrophage activation and massive cytokine secretion in cell culture and mouse xenografts. In individuals with ENKTL, ECSIT-V140A was associated with activation of NF-κB, higher HPS incidence, and poor prognosis. The immunosuppressive drug thalidomide prevented NF-κB from binding to the promoters of its target genes (including TNF and IFNG), and combination treatment with thalidomide and dexamethasone extended survival of mice engrafted with ECSIT-T419C-transfected ENKTL cells. We added thalidomide to the conventional dexamethasone-containing therapy regimen for two patients with HPS who expressed ECSIT-V140A, and we observed reversal of their HPS and disease-free survival for longer than 3 years. These findings provide mechanistic insights and a potential therapeutic strategy for ENKTL-associated HPS.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Inflammation/genetics , Lymphohistiocytosis, Hemophagocytic/genetics , Lymphoma, Extranodal NK-T-Cell/genetics , Adaptor Proteins, Signal Transducing/chemistry , Adult , Calgranulin A/chemistry , Calgranulin A/genetics , Calgranulin B/chemistry , Calgranulin B/genetics , Dexamethasone/administration & dosage , Female , Gene Knock-In Techniques , Heterografts , Humans , Inflammation/drug therapy , Inflammation/pathology , Interferon-gamma/genetics , Lymphohistiocytosis, Hemophagocytic/complications , Lymphohistiocytosis, Hemophagocytic/drug therapy , Lymphohistiocytosis, Hemophagocytic/physiopathology , Lymphoma, Extranodal NK-T-Cell/complications , Lymphoma, Extranodal NK-T-Cell/drug therapy , Lymphoma, Extranodal NK-T-Cell/physiopathology , Male , Middle Aged , Mutation , NF-kappa B/genetics , Protein Multimerization/genetics , Signal Transduction , Thalidomide/administration & dosage , Transcription Factor RelA/genetics , Tumor Necrosis Factor-alpha/genetics , Exome Sequencing
18.
Protein J ; 35(5): 363-370, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27757788

ABSTRACT

Calprotectin is member of the S-100 protein family with a wide plethora of intra-and extracellular functions. Anticancer activities, antimicrobial effects and being a qualified disease marker are among the compelling features of this protein to be used as a pharmaceutical agent. However, there are several impediments to applications of protein pharmaceuticals including: proteolytic degradation, short circulating half-life, low solubility and immunogenicity. Pegylation is a common bioconjugation polymer capable of overcoming these drawbacks. Recombinant expression and purification of calprotectin along with its pegylation would result in enhanced pharmaco-dynamic and pharmacokinetic properties. Our florescence spectroscopy and far Ultraviolet-optical density results indicate that pegylation altered the physical and structural properties of the calprotectin to become in a more stable and functionally active state. Due to enhanced pharmacodynamic and pharmacokinetic properties of the calprotectin via pegylation, this study would pave the way for better in vitro and in vivo validations of calprotectin applications in medical practice.


Subject(s)
Calgranulin A/chemistry , Calgranulin B/chemistry , Polyethylene Glycols/chemistry , Calgranulin A/genetics , Calgranulin B/genetics , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
19.
Biochim Biophys Acta ; 1864(11): 1558-69, 2016 11.
Article in English | MEDLINE | ID: mdl-27524699

ABSTRACT

Human S100A9 (Calgranulin B) is a Ca(2+)-binding protein, from the S100 family, that often presents as a homodimer in myeloid cells. It becomes an important mediator during inflammation once calcium binds to its EF-hand motifs. Human RAGE protein (receptor for advanced glycation end products) is one of the target-proteins. RAGE binds to a hydrophobic surface on S100A9. Interactions between these proteins trigger signal transduction cascades, promoting cell growth, proliferation, and tumorigenesis. Here, we present the solution structure of mutant S100A9 (C3S) homodimer, determined by multi-dimensional NMR experiments. We further characterize the solution interactions between mS100A9 and the RAGE V domain via NMR spectroscopy. CHAPS is a zwitterionic and non-denaturing molecule widely used for protein solubilizing and stabilization. We found out that CHAPS and RAGE V domain would interact with mS100A9 by using (1)H-(15)N HSQC NMR titrations. Therefore, using the HADDOCK program, we superimpose two binary complex models mS100A9-RAGE V domain and mS100A9-CHAPS and demonstrate that CHAPS molecules could play a crucial role in blocking the interaction between mS100A9 and the RAGE V domain. WST-1 assay results also support the conclusion that CHAPS inhibits the bioactivity of mS100A9. This report will help to inform new drug development against cell proliferation.


Subject(s)
Antineoplastic Agents/pharmacology , Calgranulin B/chemistry , Cell Proliferation/drug effects , Cholic Acids/pharmacology , Epithelial Cells/drug effects , Receptor for Advanced Glycation End Products/chemistry , Amino Acid Sequence , Antineoplastic Agents/chemistry , Binding Sites , Calgranulin B/genetics , Calgranulin B/metabolism , Cell Line, Tumor , Cholic Acids/chemistry , Cloning, Molecular , Epithelial Cells/metabolism , Epithelial Cells/pathology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Domains , Protein Multimerization , Protein Structure, Secondary , Receptor for Advanced Glycation End Products/antagonists & inhibitors , Receptor for Advanced Glycation End Products/genetics , Receptor for Advanced Glycation End Products/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship
20.
Mol Biol (Mosk) ; 50(1): 136-42, 2016.
Article in Russian | MEDLINE | ID: mdl-27028819

ABSTRACT

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.


Subject(s)
Calcium/pharmacology , Calgranulin A/chemistry , Calgranulin B/chemistry , Recombinant Proteins/chemistry , Calgranulin A/genetics , Calgranulin A/isolation & purification , Calgranulin B/genetics , Calgranulin B/isolation & purification , Escherichia coli/genetics , Protein Stability/drug effects , Protein Structure, Secondary/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
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