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1.
J Exp Med ; 216(10): 2348-2361, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31337736

RESUMO

Both α-tryptase and ß-tryptase are preferentially expressed by human mast cells, but the purpose of α-tryptase is enigmatic, because its tetramers lack protease activity, whereas ß-tryptase tetramers are active proteases. The monogenic disorder called hereditary α-tryptasemia, due to increased α-tryptase gene copies and protein expression, presents with clinical features such as vibratory urticaria and dysautonomia. We show that heterotetramers composed of 2α- and 2ß-tryptase protomers (α/ß-tryptase) form naturally in individuals who express α-tryptase. α/ß-Tryptase, but not homotetramer, activates protease-activated receptor-2 (PAR2), which is expressed on cell types such as smooth muscle, neurons, and endothelium. Also, only α/ß-tryptase makes mast cells susceptible to vibration-triggered degranulation by cleaving the α subunit of the EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) mechanosensory receptor. Allosteric effects of α-tryptase protomers on neighboring ß-tryptase protomers likely result in the novel substrate repertoire of α/ß-tryptase tetramers that in turn cause some of the clinical features of hereditary α-tryptasemia and of other disorders involving mast cells.

2.
J Allergy Clin Immunol ; 141(1): 311-321.e10, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28624612

RESUMO

BACKGROUND: Mast cells (MCs), the primary effector cell of the atopic response, participate in immune defense at host/environment interfaces, yet the mechanisms by which they interact with CD4+ T cells has been controversial. OBJECTIVE: We used in situ-matured primary human MCs and matched CD4+ T cells to diligently assess the ability of MCs to act as antigen-presenting cells. METHODS: We examined mature human skin-derived MCs using flow cytometry for expression of antigen-presenting molecules, for their ability to stimulate CD4+ T cells to express CD25 and proliferate when exposed to superantigen or to cytomegalovirus (CMV) antigen using matched T cells and MCs from CMV-seropositive or CMV-seronegative donors, and for antigen uptake. Subcellular localization of antigen, HLA molecules, and tryptase was analyzed by using structured illumination microscopy. RESULTS: Our data show that IFN-γ induces HLA class II, HLA-DM, CD80, and CD40 expression on MCs, whereas MCs take up soluble and particulate antigens in an IFN-γ-independent manner. IFN-γ-primed MCs guide activation of T cells by Staphylococcus aureus superantigen and, when preincubated with CMV antigens, induce a recall CD4+ TH1 proliferation response only in CMV-seropositive donors. MCs co-opt their secretory granules for antigen processing and presentation. Consequently, MC degranulation increases surface delivery of HLA class II/peptide, further enhancing stimulation of T-cell proliferation. CONCLUSIONS: IFN-γ primes human MCs to activate T cells through superantigen and to present CMV antigen to TH1 cells, co-opting MC secretory granules for antigen processing and presentation and creating a feed-forward loop of T-cell-MC cross-activation.


Assuntos
Apresentação do Antígeno , Linfócitos T CD4-Positivos/imunologia , Mastócitos/imunologia , Apresentação do Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos Virais/imunologia , Transporte Biológico , Biomarcadores , Linfócitos T CD4-Positivos/metabolismo , Comunicação Celular , Células Cultivadas , Dinaminas , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Mastócitos/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T
3.
Nat Genet ; 48(12): 1564-1569, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27749843

RESUMO

Elevated basal serum tryptase levels are present in 4-6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase-encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.


Assuntos
Dor Crônica/genética , Doenças do Tecido Conjuntivo/genética , Variações do Número de Cópias de DNA/genética , Disautonomia Familiar/genética , Gastroenteropatias/genética , Prurido/genética , Dermatopatias/genética , Triptases/sangue , Triptases/genética , Adolescente , Adulto , Idoso , Criança , Dor Crônica/sangue , Dor Crônica/enzimologia , Doenças do Tecido Conjuntivo/sangue , Doenças do Tecido Conjuntivo/enzimologia , Disautonomia Familiar/sangue , Disautonomia Familiar/enzimologia , Feminino , Gastroenteropatias/sangue , Gastroenteropatias/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Prurido/sangue , Prurido/enzimologia , Dermatopatias/sangue , Dermatopatias/enzimologia , Adulto Jovem
4.
Sci Rep ; 6: 25670, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27181495

RESUMO

Moiré superlattices in graphene supported on various substrates have opened a new avenue to engineer graphene's electronic properties. Yet, the exact crystallographic structure on which their band structure depends remains highly debated. In this scanning tunneling microscopy and density functional theory study, we have analysed graphene samples grown on multilayer graphene prepared onto SiC and on the close-packed surfaces of Re and Ir with ultra-high precision. We resolve small-angle twists and shears in graphene, and identify large unit cells comprising more than 1,000 carbon atoms and exhibiting non-trivial nanopatterns for moiré superlattices, which are commensurate to the graphene lattice. Finally, a general formalism applicable to any hexagonal moiré is presented to classify all reported structures.

5.
J Immunol ; 187(4): 1912-8, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21742978

RESUMO

Human ß-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. ß-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human ß-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature ß-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.


Assuntos
Catepsina B/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Precursores Enzimáticos/metabolismo , Mastócitos/enzimologia , Triptases/metabolismo , Animais , Catepsina B/genética , Catepsina B/imunologia , Catepsina C/genética , Catepsina C/imunologia , Catepsina L/genética , Catepsina L/imunologia , Linhagem Celular Tumoral , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Humanos , Mastócitos/imunologia , Camundongos , Camundongos Mutantes , Vesículas Secretórias/enzimologia , Vesículas Secretórias/genética , Vesículas Secretórias/imunologia , Pele/enzimologia , Pele/imunologia , Triptases/genética , Triptases/imunologia
6.
J Immunol ; 186(12): 7136-43, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21562164

RESUMO

Human α- and ß-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. ß-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. ß-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated ß-protryptase (R(-3)Q) as well as wild-type ß-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and ß-protryptases from human mast cells to their mature enzymatically active products.


Assuntos
Catepsinas/metabolismo , Precursores Enzimáticos/metabolismo , Mastócitos/enzimologia , Processamento de Proteína Pós-Traducional , Triptases/metabolismo , Catepsina B/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Catepsinas/análise , Linhagem Celular , Heparina/farmacologia , Humanos , Espectrometria de Massas , Mastócitos/metabolismo
7.
Electrophoresis ; 26(6): 1038-45, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15669004

RESUMO

A novel, sensitive method for detecting protease inhibitors by using fluorescent protease substrates in gels is described. The protease inhibitors were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels containing a copolymerized peptide substrate, namely 4-methyl-coumaryl-7-amide (MCA). As the incorporated substrates in the gel, Boc-Phe Ser-Arg-MCA was used for trypsin, Suc-Ala-Ala-Pro-Phe-MCA for alpha-chymotrypsin, and Z-Phe-Arg-MCA for papain. After electrophoresis, washing and incubating the gel with the target protease solutions allowed the substrate to be cleaved by the protease, and the release of the fluorescent 7 amino-4 methyl-coumarin (AMC), which was detected under a UV transilluminator. The uncleaved peptide-MCA substrate remained where the inhibitors were present, and was visualized as dark blue bands on the light-green fluorescent background gel. This new method offers several advantages over other previous methods including: (i) greatly increased sensitivity can be achieved in a shorter period of time, which may be useful for discovering new protease inhibitors in small amounts of crude material; (ii) the procedure is quite simple and quick since the incubation period is very short and no time is needed for staining and destaining steps; (iii) since these probes using substrate specificity/target proteases, they are excellent tools for detection and discrimination of unknown protease inhibitors for various target proteases.


Assuntos
Cumarínicos/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes , Inibidores de Proteases/isolamento & purificação , Animais , Quimotripsina/antagonistas & inibidores , Mucosa Intestinal/química , Intestino Delgado/química , Sensibilidade e Especificidade , Suínos , Inibidores da Tripsina/isolamento & purificação
8.
Adv Enzyme Regul ; 44: 1-10, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15581478

RESUMO

We found a novel procaspase-3 activating cascade mediated by lysosomal enzyme. The activating enzyme of procaspase-3, named lysoapoptase having the molecular weight of 78kDa was determined to be a lactoferrin located in the lysosome. Recombinant lactoferrin accelerated the processing of procaspase-3 to form active caspase-3 in vitro. D-Galactosamine is a well-known inducer of hepatocyte apoptosis. The caspase-3 which plays a common central role in the final step of various apoptosis cascades, was dramatically increased in the cytoplasm by the d-galactosamine administration in vivo. When D-galactosamine was administrated as a death signal in vivo, the lysosomal lactoferrin was released into the cytoplasm and procaspase-3 located in the cytoplasm was processed to form active caspase-3. The cotreatment of epigallo-catechin gallate resulted in the complete protection of the hepatocyte apoptosis suppressing the increases of caspase-3 in the cytoplasm. The caspase-3 activity was also inhibited directly by the epigallo-catechin gallate. Therefore, all apoptosis cascades mediated by caspase-3 should be suppressed by epigallo-catechin gallate. The caspase-3 activity was not only directly inhibited by epigallo-catechin gallate in vitro, but the release of lactoferrin from the lysosomes into the cytoplasm was also suppressed by epigallo-catechin gallate treatment in vivo. Therefore, the apoptosis induction was suppressed at the two successive steps by cotreatment of epigallo-catechin gallate in vivo.


Assuntos
Apoptose/fisiologia , Catequina/análogos & derivados , Catequina/farmacologia , Lisossomos/enzimologia , Inibidores de Proteases/farmacologia , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/isolamento & purificação , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Galactosamina/toxicidade , Cinética , Lactoferrina/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo
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