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1.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34207949

RESUMO

BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.


Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Anticorpos de Domínio Único/química , Microglobulina beta-2/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Elementos Estruturais de Proteínas , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Microglobulina beta-2/imunologia
2.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199259

RESUMO

Beta2-microglobulin (B2M) a key component of major histocompatibility complex class I molecules, which aid cytotoxic T-lymphocyte (CTL) immune response. However, the majority of studies of B2M have focused only on amyloid fibrils in pathogenesis to the neglect of its role of antimicrobial activity. Indeed, B2M also plays an important role in innate defense and does not only function as an adjuvant for CTL response. A previous study discovered that human aggregated B2M binds the surface protein structure in Streptococci, and a similar study revealed that sB2M-9, derived from native B2M, functions as an antibacterial chemokine that binds Staphylococcus aureus. An investigation of sB2M-9 exhibiting an early lymphocyte recruitment in the human respiratory epithelium with bacterial challenge may uncover previously unrecognized aspects of B2M in the body's innate defense against Mycobactrium tuberculosis. B2M possesses antimicrobial activity that operates primarily under pH-dependent acidic conditions at which B2M and fragmented B2M may become a nucleus seed that triggers self-aggregation into distinct states, such as oligomers and amyloid fibrils. Modified B2M can act as an antimicrobial peptide (AMP) against a wide range of microbes. Specifically, these AMPs disrupt microbe membranes, a feature similar to that of amyloid fibril mediated cytotoxicity toward eukaryotes. This study investigated two similar but nonidentical effects of B2M: the physiological role of B2M, in which it potentially acts against microbes in innate defense and the role of B2M in amyloid fibrils, in which it disrupts the membrane of pathological cells. Moreover, we explored the pH-governing antibacterial activity of B2M and acidic pH mediated B2M amyloid fibrils underlying such cytotoxicity.


Assuntos
Amiloide/toxicidade , Antibacterianos/farmacologia , Microglobulina beta-2/metabolismo , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Microglobulina beta-2/química
3.
RNA ; 27(10): 1241-1256, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34244458

RESUMO

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.


Assuntos
Grânulos Citoplasmáticos/genética , Retículo Endoplasmático/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Resposta a Proteínas não Dobradas , Biomarcadores/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dactinomicina/farmacologia , Diterpenos/farmacologia , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Compostos de Epóxi/farmacologia , Expressão Gênica , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Biogênese de Organelas , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fenantrenos/farmacologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Imagem Individual de Molécula , Estresse Fisiológico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
4.
Immunity ; 54(8): 1772-1787.e9, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34289378

RESUMO

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.


Assuntos
Amiloide/metabolismo , Mieloma Múltiplo/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos Associados a Tumor/metabolismo , Microglobulina beta-2/metabolismo , Animais , Células Cultivadas , Humanos , Inflamação/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lisossomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fagocitose/imunologia , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Microglobulina beta-2/genética
5.
Int J Mol Sci ; 22(9)2021 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-34063223

RESUMO

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.


Assuntos
Amiloide/metabolismo , Tripsina/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Naftalenossulfonato de Anilina , Benzotiazóis , Corantes Fluorescentes , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Muramidase/metabolismo , Proteólise , Tripsina/química , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo
6.
Genes (Basel) ; 12(4)2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919966

RESUMO

The bottlenose dolphin (Tursiops truncatus) belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T. truncatus T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3' end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.


Assuntos
Golfinho Nariz-de-Garrafa/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de Proteína/métodos , Animais , Mapeamento Cromossômico , Feminino , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Alinhamento de Sequência , Microglobulina beta-2/metabolismo
7.
Exp Gerontol ; 150: 111359, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33905876

RESUMO

Beta 2 microglobulin (Β2M) is expressed in all nucleated cells, it interplays with mediators to regulate and modulate cellular functions. Its role in aging associated disorders has been documented recently. Oxidative stress has been known to play a direct implication on these disorders. Therefore, there is a rationality to explore the function of Β2M in oxidative stress in elderly people. The aim of the study was to assess the Β2M levels in different group of age, and to study the correlation between Β2M and oxidative stress. Actually, the serum levels of Β2M increased significantly in old people comparing to youngers. In addition, there was a positive correlation between Β2M levels and the age of participants (p < 0.001). In addition, there was a positive correlation between Β2M levels and Malondialdehyde (MDA) (p < 0.001), which underscored the possible role of Β2M in oxidative stress. To confirm the previous result, the correlation between total antioxidant capacity (TAC) and Β2M was assessed. There was a negative correlation between them (p < 0.001). These results suggested a possible role of Β2M in oxidative stress status in elderly people; in addition, it suggested the ability of using Β2M as a novel biomarker for oxidative stress. However, further work should be conducted to explore the exact role of Β2M in oxidative stress, and to include large sample size to confirm the results before translating the findings to clinic.


Assuntos
Estresse Oxidativo , Microglobulina beta-2 , Idoso , Biomarcadores , Humanos , Microglobulina beta-2/metabolismo
8.
J Vis Exp ; (169)2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33779591

RESUMO

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous ß2-microglobulin (ß2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous ß2m is not available. Meanwhile, it is indicated that mammalian ß2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous ß2-microglobulin (ß2m). Herein, methods to evaluate the feasibility of heterologous ß2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Microglobulina beta-2/metabolismo , Animais , Quirópteros
9.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467468

RESUMO

The bottom-up design of smart nanodevices largely depends on the accuracy by which each of the inherent nanometric components can be functionally designed with predictive methods. Here, we present a rationally designed, self-assembled nanochip capable of capturing a target protein by means of pre-selected binding sites. The sensing elements comprise computationally evolved peptides, designed to target an arbitrarily selected binding site on the surface of beta-2-Microglobulin (ß2m), a globular protein that lacks well-defined pockets. The nanopatterned surface was generated by an atomic force microscopy (AFM)-based, tip force-driven nanolithography technique termed nanografting to construct laterally confined self-assembled nanopatches of single stranded (ss)DNA. These were subsequently associated with an ssDNA-peptide conjugate by means of DNA-directed immobilization, therefore allowing control of the peptide's spatial orientation. We characterized the sensitivity of such peptide-containing systems against ß2m in solution by means of AFM-based differential topographic imaging and surface plasmon resonance (SPR) spectroscopy. Our results show that the confined peptides are capable of specifically capturing ß2m from the surface-liquid interface with micromolar affinity, hence providing a viable proof-of-concept for our approach to peptide design.


Assuntos
Biologia Computacional/métodos , DNA de Cadeia Simples/metabolismo , Peptídeos/metabolismo , Microglobulina beta-2/metabolismo , Sítios de Ligação/genética , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Humanos , Cinética , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Ressonância de Plasmônio de Superfície/métodos , Microglobulina beta-2/química , Microglobulina beta-2/genética
10.
Commun Biol ; 4(1): 146, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514846

RESUMO

The efficiency of the repair process following ischemic cardiac injury is a crucial determinant for the progression into heart failure and is controlled by both intra- and intercellular signaling within the heart. An enhanced understanding of this complex interplay will enable better exploitation of these mechanisms for therapeutic use. We used single-cell transcriptomics to collect gene expression data of all main cardiac cell types at different time-points after ischemic injury. These data unveiled cellular and transcriptional heterogeneity and changes in cellular function during cardiac remodeling. Furthermore, we established potential intercellular communication networks after ischemic injury. Follow up experiments confirmed that cardiomyocytes express and secrete elevated levels of beta-2 microglobulin in response to ischemic damage, which can activate fibroblasts in a paracrine manner. Collectively, our data indicate phase-specific changes in cellular heterogeneity during different stages of cardiac remodeling and allow for the identification of therapeutic targets relevant for cardiac repair.


Assuntos
Perfilação da Expressão Gênica , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/metabolismo , Análise de Célula Única , Transcriptoma , Remodelação Ventricular , Cicatrização , Microglobulina beta-2/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Comunicação Parácrina , Fatores de Tempo , Microglobulina beta-2/metabolismo
11.
Circ Res ; 128(5): 655-669, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33508948

RESUMO

RATIONALE: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived ß2M (ß-2 microglobulin) and TGF-ß (transforming growth factor ß) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. OBJECTIVE: To determine the molecular mechanisms and signal transduction pathways by which ß2M and TGF-ß regulate monocyte responses both in vitro and in vivo. METHODS AND RESULTS: Wild-type- (WT) and platelet-specific ß2M knockout mice were treated intravenously with either ß2M or TGF-ß to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma ß2M increased proinflammatory monocytes, while increased plasma TGFß increased proreparative monocytes. TGF-ßR (TGF-ß receptor) inhibition blunted monocyte responses to both ß2M and TGF-ß in vivo. Using imaging flow cytometry, we found that ß2M decreased monocyte SMAD2/3 nuclear localization, while TGF-ß promoted SMAD nuclear translocation but decreased noncanonical/inflammatory (JNK [jun kinase] and NF-κB [nuclear factor-κB] nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. ß2M, but not TGF-ß, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a proreparative monocyte response. CONCLUSIONS: Our findings indicate that elevated plasma ß2M and TGF-ß dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor but induce SMAD-dependent canonical signaling (TGF-ß) versus noncanonical SMAD-independent signaling (ß2M) in a ubiquitin ligase dependent manner. This work has broad implications as ß2M is increased in several inflammatory conditions, while TGF-ß is increased in fibrotic diseases. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Monócitos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Microglobulina beta-2/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Smad/metabolismo , Células THP-1 , Microglobulina beta-2/farmacologia
12.
Am J Kidney Dis ; 77(5): 673-683.e1, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33301877

RESUMO

RATIONALE AND OBJECTIVE: Glomerular filtration rate (GFR) estimation based on creatinine and cystatin C (eGFRcr-cys) is more accurate than estimated GFR (eGFR) based on creatinine or cystatin C alone (eGFRcr or eGFRcys, respectively), but the inclusion of creatinine in eGFRcr-cys requires specification of a person's race. ß2-Microglobulin (B2M) and ß-trace protein (BTP) are alternative filtration markers that appear to be less influenced by race than creatinine is. STUDY DESIGN: Study of diagnostic test accuracy. SETTING AND PARTICIPANTS: Development in a pooled population of 7 studies with 5,017 participants with and without chronic kidney disease. External validation in a pooled population of 7 other studies with 2,245 participants. TESTS COMPARED: Panel eGFR using B2M and BTP in addition to cystatin C (3-marker panel) or creatinine and cystatin C (4-marker panel) with and without age and sex or race. OUTCOMES: GFR measured as the urinary clearance of iothalamate, plasma clearance of iohexol, or plasma clearance of [51Cr]EDTA. RESULTS: Mean measured GFRs were 58.1 and 83.2 mL/min/1.73 m2, and the proportions of Black participants were 38.6% and 24.0%, in the development and validation populations, respectively. In development, addition of age and sex improved the performance of all equations compared with equations without age and sex, but addition of race did not further improve the performance. In validation, the 4-marker panels were more accurate than the 3-marker panels (P < 0.001). The 3-marker panel without race was more accurate than eGFRcys (percentage of estimates greater than 30% different from measured GFR [1 - P30] of 15.6% vs 17.4%; P = 0.01), and the 4-marker panel without race was as accurate as eGFRcr-cys (1 - P30 of 8.6% vs 9.4%; P = 0.2). Results were generally consistent across subgroups. LIMITATIONS: No representation of participants with severe comorbid illness and from geographic areas outside of North America and Europe. CONCLUSIONS: The 4-marker panel eGFR is as accurate as eGFRcr-cys without requiring specification of race. A more accurate race-free eGFR could be an important advance.


Assuntos
Afro-Americanos , Creatinina/metabolismo , Cistatina C/metabolismo , Grupo com Ancestrais do Continente Europeu , Taxa de Filtração Glomerular , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Insuficiência Renal Crônica/diagnóstico , Microglobulina beta-2/metabolismo , Adolescente , Adulto , Grupo com Ancestrais do Continente Africano , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Radioisótopos de Cromo , Ácido Edético , Feminino , Humanos , Iohexol , Ácido Iotalâmico , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/etnologia , Insuficiência Renal Crônica/metabolismo , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Adulto Jovem
13.
Mol Neurobiol ; 58(1): 243-262, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32918239

RESUMO

Mitochondrial diseases (MD), such as Leigh syndrome (LS), present with severe neurological and muscular phenotypes in patients, but have no known cure and limited treatment options. Based on their neuroprotective effects against other neurodegenerative diseases in vivo and their positive impact as an antioxidant against complex I deficiency in vitro, we investigated the potential protective effect of metallothioneins (MTs) in an Ndufs4 knockout mouse model (with a very similar phenotype to LS) crossed with an Mt1 overexpressing mouse model (TgMt1). Despite subtle reductions in the expression of neuroinflammatory markers GFAP and IBA1 in the vestibular nucleus and hippocampus, we found no improvement in survival, growth, locomotor activity, balance, or motor coordination in the Mt1 overexpressing Ndufs4-/- mice. Furthermore, at a cellular level, no differences were detected in the metabolomics profile or gene expression of selected one-carbon metabolism and oxidative stress genes, performed in the brain and quadriceps, nor in the ROS levels of macrophages derived from these mice. Considering these outcomes, we conclude that MT1, in general, does not protect against the impaired motor activity or improve survival in these complex I-deficient mice. The unexpected absence of increased oxidative stress and metabolic redox imbalance in this MD model may explain these observations. However, tissue-specific observations such as the mildly reduced inflammation in the hippocampus and vestibular nucleus, as well as differential MT1 expression in these tissues, may yet reveal a tissue- or cell-specific role for MTs in these mice.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Metalotioneína/metabolismo , Doenças Mitocondriais/patologia , Doenças Mitocondriais/prevenção & controle , Animais , Ataxia/complicações , Ataxia/patologia , Ataxia/fisiopatologia , Biomarcadores/metabolismo , Peso Corporal , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Hipocampo/patologia , Inflamação/sangue , Inflamação/patologia , Masculino , Metaboloma , Metalotioneína/genética , Camundongos Knockout , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , Atividade Motora , Oxirredução , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Microglobulina beta-2/metabolismo
14.
J Immunol ; 205(11): 3095-3106, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33148716

RESUMO

Iron is an essential element for Mycobacterium tuberculosis; it has at least 40 enzymes that require iron as a cofactor. Accessibility of iron at the phagosomal surface inside macrophage is crucial for survival and virulence of M. tuberculosis ESAT-6, a 6-kDa-secreted protein of region of difference 1, is known to play a crucial role in virulence and pathogenesis of M. tuberculosis In our earlier study, we demonstrated that ESAT-6 protein interacts with ß-2-microglobulin (ß2M) and affects class I Ag presentation through sequestration of ß2M inside endoplasmic reticulum, which contributes toward inhibition of MHC class I:ß2M:peptide complex formation. The 6 aa at C-terminal region of ESAT-6 are essential for ESAT6:ß2M interaction. ß2M is essential for proper folding of HFE, CD1, and MHC class I and their surface expression. It is known that M. tuberculosis recruit holotransferrin at the surface of the phagosome. But the upstream mechanism by which it modulates holotransferrin-mediated iron uptake at the surface of macrophage is not well understood. In the current study, we report that interaction of the ESAT-6 protein with ß2M causes downregulation of surface HFE, a protein regulating iron homeostasis via interacting with transferrin receptor 1 (TFR1). We found that ESAT-6:ß2M interaction leads to sequestration of HFE in endoplasmic reticulum, causing poorer surface expression of HFE and HFE:TFR1 complex (nonfunctional TFR1) in peritoneal macrophages from C57BL/6 mice, resulting in increased holotransferrin-mediated iron uptake in these macrophages. These studies suggest that M. tuberculosis probably targets the ESAT-6 protein to increase iron uptake.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Regulação para Baixo/fisiologia , Proteína da Hemocromatose/metabolismo , Macrófagos Peritoneais/metabolismo , Mycobacterium tuberculosis/metabolismo , Transferrina/metabolismo , Animais , Transporte Biológico/fisiologia , Retículo Endoplasmático/metabolismo , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores da Transferrina/metabolismo , Virulência/fisiologia , Microglobulina beta-2/metabolismo
15.
Int J Med Sci ; 17(18): 3058-3064, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33173426

RESUMO

Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene (BDNF). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning. Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF. Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) - relative to housekeeping genes - and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR). Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion: Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Alelos , Biópsia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Treino Aeróbico , Teste de Esforço/métodos , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Voluntários Saudáveis , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Masculino , Metionina/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Músculo Quadríceps/patologia , Valina/genética , Adulto Jovem , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
16.
J Am Chem Soc ; 142(49): 20845-20854, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33253560

RESUMO

Protein-protein interactions (PPIs) are involved in many of life's essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein ß2-microglobulin (ß2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped, off-pathway oligomers using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of "post-translational chemical modification" as a tool to study biological molecular mechanisms.


Assuntos
Proteínas Amiloidogênicas/química , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Cristalografia por Raios X , Dissulfetos/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo
17.
J Control Release ; 327: 8-18, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745567

RESUMO

Metabolic pathways in the body are highly specific. Dysfunction of a metabolic pathway triggers the accumulation of its target substance. For example, kidney failure results in increased ß2-microglobulin blood levels, causing dialysis-related amyloidosis. Previously, we proposed a novel therapeutic concept, that is a removal of an etiologic factor of metabolic disease by artificial switching of its metabolic processing pathway, and tested this concept using in cultured cells. However, the feasibility of artificial metabolic switching in vivo remained unknown. Here, we show that a newly developed "navigator" molecule changes the metabolic processing pathway of ß2-microglobulin from the kidney to the liver in mouse. The artificial metabolic switching is achieved by the capture of the etiologic factor by the navigator, which then steers the etiologic factor to hepatic lysosomes via low-density lipoprotein receptors. These findings demonstrate that navigator-based artificial metabolic switching can be a therapeutic strategy for various diseases caused by metabolic disorders.


Assuntos
Amiloidose , Diálise Renal , Animais , Redes e Vias Metabólicas , Camundongos , Receptores de LDL , Microglobulina beta-2/metabolismo
18.
Drug Metab Dispos ; 48(10): 925-933, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32723849

RESUMO

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and ß2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and ß2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and ß2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic ß2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and ß2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and ß2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.


Assuntos
Produtos Biológicos/farmacocinética , Antígenos de Histocompatibilidade Classe I/análise , Fígado/metabolismo , Modelos Biológicos , Receptores Fc/análise , Microglobulina beta-2/análise , Adulto , Produtos Biológicos/administração & dosagem , Variação Biológica da População , Cromatografia Líquida de Alta Pressão/métodos , Relação Dose-Resposta a Droga , Eliminação Hepatobiliar , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Proteômica/métodos , Receptores Fc/metabolismo , Adulto Jovem , Microglobulina beta-2/metabolismo
19.
BMC Res Notes ; 13(1): 294, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32552881

RESUMO

OBJECTIVES: ß2 microglobulin, as a biomarker, is used for the diagnosis of oral malignant and pre-malignant lesions. The components of the microglobulin system can directly or indirectly help grow and develop tumors. The present study aims to compare beta-2 microglobulin levels in patients with lichen planus of the esophagus, oral squamous cell carcinoma, and healthy individuals. Further, it evaluated the salivary ß2-microglobulin level in malignant and pre-malignant lesions. Oral lichen planus (OLP) is a chronic skin-mucus disorder. Of the total 75 patients referred to Oral Medicine at Dentistry School of Zahedan University of Medical Sciences, 25 were healthy and 25 had oral lichen planus (OLP) and the rest had squamous cell carcinoma (SCC). To collect the saliva samples, unstimulated spitting was used. They were collected between 9 and 12 a.m. Salivary beta2 microglobulin was recorded based on the factory instructions by ELISA optical density method with 450 nm wavelength for each sample. The data were analyzed using descriptive, Kruskal-Wallis and Mann-Whitney and Pearson's correlation coefficient (SPSS 21). RESULTS: The salivary ß2 microglobulin level in patients with squamous cell carcinoma (SCC) and oral lichen plan (OLP) is significantly higher than that in healthy group. Thus, this index is used for assessing early malignant transformation and oral pre-malignant lesion.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Transformação Celular Neoplásica/metabolismo , Líquen Plano Bucal/metabolismo , Neoplasias Bucais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Microglobulina beta-2/metabolismo , Adulto , Carcinoma de Células Escamosas/diagnóstico , Estudos Transversais , Esôfago/patologia , Humanos , Líquen Plano Bucal/diagnóstico , Neoplasias Bucais/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Saliva/metabolismo
20.
Hematol Oncol ; 38(4): 493-500, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32533716

RESUMO

The aim was to build a prognostic model to stratify patients at diagnosis into different risk categories. We investigated the prognostic value of functional PET parameters and clinical features in 64 primary breast lymphoma (PBL) patients. With a median follow-up of 60 months, 5-year progression-free survival (PFS) and overall survival (OS) was 62.5% and 73.4%. In multivariate analysis, baseline total metabolic tumor volume (TMTV0) and ß2-microglobulin remained more reliable predictors of survival than other prognostic factors. The optimal TMTV0 cut-off value was 90 cm3 . Among 29 patients with high TMTV0, 5-year PFS and OS were 44.8% and 62.1%, respectively, while 5-year PFS and OS of 35 patients with low TMTV0 were 74.3% and 85.7%, respectively. TMTV0 combined with ß2-microglobulin identified three groups with very different prognosis, including low-risk group with low TMTV0 and ß2-microglobulin≤normal (n = 30), intermediate-risk group with high TMTV0 or ß2-microglobulin>normal (n = 20), and high-risk group with high TMTV0 and ß2-microglobulin>normal (n = 14). In the three groups, 5-year PFS rates were 80%, 55% and 28.6% (P = .003), and 5-year OS rates were 90%, 65%, and 50% (P = .023) respectively. We established a new prognostic model through TMTV0 and ß2-microglobulin, and can divide PBL at diagnosis into different risk categories.


Assuntos
Neoplasias da Mama/patologia , Fluordesoxiglucose F18/metabolismo , Linfoma/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Carga Tumoral , Microglobulina beta-2/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Terapia Combinada , Feminino , Seguimentos , Humanos , Linfoma/diagnóstico por imagem , Linfoma/metabolismo , Linfoma/terapia , Pessoa de Meia-Idade , Prognóstico , Compostos Radiofarmacêuticos/metabolismo , Taxa de Sobrevida , Adulto Jovem
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