RESUMO
As central effectors of the adaptive immune response, immunoglobulins, or antibodies, provide essential protection from pathogens through their ability to recognize foreign antigens, aid in neutralization, and facilitate elimination from the host. Mammalian immunoglobulins can be classified into five isotypes-IgA, IgD, IgE, IgG, and IgM-each with distinct roles in mediating various aspects of the immune response. Of these isotypes, IgA and IgM are the only ones capable of multimerization, arming them with unique biological functions. Increased valency of polymeric IgA and IgM provides high avidity for binding low-affinity antigens, and their ability to be transported across the mucosal epithelium into secretions by the polymeric immunoglobulin receptor allows them to play critical roles in mucosal immunity. Here we discuss the molecular assembly, structure, and function of these multimeric antibodies.
Assuntos
Imunoglobulina A , Receptores de Imunoglobulina Polimérica , Animais , Humanos , Imunidade nas Mucosas , Imunoglobulina M/química , Imunoglobulina M/metabolismo , Mamíferos/metabolismo , Mucosa , Receptores de Imunoglobulina Polimérica/químicaRESUMO
RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação V(D)J , Animais , Linhagem Celular , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese , Sinais Direcionadores de Proteínas , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de ProteínaRESUMO
The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.
Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Neoplasias do Colo do Útero/enzimologia , DNA de Neoplasias/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Feminino , Células HEK293 , Células HeLa , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Enhancers are the key regulators of other DNA-based processes by virtue of their unique ability to generate nucleosome-depleted regions in a highly regulated manner. Enhancers regulate cell-type-specific transcription of tRNA genes by RNA polymerase III (Pol III). They are also responsible for the binding of the origin replication complex (ORC) to DNA replication origins, thereby regulating origin utilization, replication timing, and replication-dependent chromosome breaks. Additionally, enhancers regulate V(D)J recombination by increasing access of the recombination-activating gene (RAG) recombinase to target sites and by generating non-coding enhancer RNAs and localized regions of trimethylated histone H3-K4 recognized by the RAG2 PHD domain. Thus, enhancers represent the first step in decoding the genome, and hence they regulate biological processes that, unlike RNA polymerase II (Pol II) transcription, do not have dedicated regulatory proteins.
Assuntos
Replicação do DNA , Elementos Facilitadores Genéticos , RNA Polimerase III , Transcrição Gênica , Recombinação V(D)J , Animais , Humanos , Replicação do DNA/genética , Regulação da Expressão Gênica/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Transcrição Gênica/genética , Recombinação V(D)J/genéticaRESUMO
V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.
Assuntos
Instabilidade Genômica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Recombinação V(D)J/genética , Animais , Sequência de Bases/genética , Células COS , Chlorocebus aethiops , Cromossomos/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Células NIH 3T3 , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Recombinases/genéticaRESUMO
The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.
Assuntos
Diversidade de Anticorpos , Núcleo Celular/imunologia , Elementos Facilitadores Genéticos , Rearranjo Gênico do Linfócito B , Cadeias kappa de Imunoglobulina/imunologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Forma do Núcleo Celular , Montagem e Desmontagem da Cromatina , Genótipo , Células HEK293 , Humanos , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Células Precursoras de Linfócitos B/metabolismo , Conformação Proteica , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Relação Estrutura-AtividadeRESUMO
Cell-cell fusion mediated by most paramyxovirus requires fusion protein (F) and attachment protein (H, HN, or G). The F protein is proteolytic cleaved to be fusogenically active. J paramyxovirus (JPV) has a unique feature in the family Paramyxoviridae: It encodes an integral membrane protein, syncytial protein (SP, formerly known as transmembrane protein, TM), which is essential in JPV-promoted cell-cell fusion (i.e., syncytial). In this study, we report that cleavage of SP is essential for its syncytial-promoting activity. We have identified the cleavage site of SP at amino acid residues 172 to 175, LKTG, and deletion of the "LKTG" residues abolished SP protein cleavage and its ability to promote cell-cell fusion. Replacing the cleavage site LKTG with a factor Xa protease cleavage site allows cleavage of the SP with factor Xa protease and restores its ability to promote cell-cell fusion. Furthermore, results from a hemifusion assay indicate that cleavage of SP plays an important role in the progression from the intermediate hemifusion state to a complete fusion. This work indicates that SP has many characteristics of a fusion protein. We propose that SP is likely a cell-cell fusion-promoting protein.
Assuntos
Fusão Celular , Proteínas Virais de Fusão , Animais , Proteínas Virais de Fusão/metabolismo , Chlorocebus aethiops , Proteólise , Células Vero , Internalização do Vírus , Fator Xa/metabolismo , Humanos , Linhagem CelularRESUMO
Deprotonation or suppression of the pKa of the amino group of a lysine sidechain is a widely recognized phenomenon whereby the sidechain amino group transiently can act as a nucleophile at the active site of enzymatic reactions. However, a deprotonated lysine and its molecular interactions have not been directly experimentally detected. Here, we demonstrate a deprotonated lysine stably serving as an "acceptor" in a H-bond between the photosensor protein RcaE and its chromophore. Signal splitting and trans-H-bond J coupling observed by NMR spectroscopy provide direct evidence that Lys261 is deprotonated and serves as a H-bond acceptor for the chromophore NH group. Quantum mechanical/molecular mechanical calculations also indicate that this H-bond exists stably. Interestingly, the sidechain amino group of the lysine can act as both donor and acceptor. The remarkable shift in the H-bond characteristics arises from a decrease in solvation, triggered by photoisomerization. Our results provide insights into the dual role of this lysine. This mechanism has broad implications for other biological reactions in which lysine plays a role.
Assuntos
Ligação de Hidrogênio , Lisina , Lisina/química , Lisina/metabolismo , Prótons , Modelos Moleculares , Espectroscopia de Ressonância MagnéticaRESUMO
The DNA double-strand break (DSB) repair factor 53BP1 has long been implicated in V(D)J and class switch recombination (CSR) of mammalian lymphocyte receptors. However, the dissection of the underlying molecular activities is hampered by a paucity of studies [V(D)J] and plurality of phenotypes (CSR) associated with 53BP1 deficiency. Here, we revisit the currently accepted roles of 53BP1 in antibody diversification in view of the recent identification of its downstream effectors in DSB protection and latest advances in genome architecture. We propose that, in addition to end protection, 53BP1-mediated end-tethering stabilization is essential for CSR. Furthermore, we support a pre-DSB role during V(D)J recombination. Our perspective underscores the importance of evaluating repair of DSBs in relation to their dynamic architectural contexts.
Assuntos
Anticorpos , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Anticorpos/genética , Switching de Imunoglobulina/genética , Linfócitos , MamíferosRESUMO
The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA Ligase Dependente de ATP/genética , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku/genética , CamundongosRESUMO
The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf -/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf -/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQRXlf -/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.
Assuntos
Proteínas Quinases , Processamento de Proteína Pós-Traducional , Animais , Camundongos , Fosforilação , Alanina , Linfócitos B , Proteína Quinase Ativada por DNA , Mamíferos , Proteínas de Ligação a DNARESUMO
Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.
Assuntos
Sequências Reguladoras de Ácido Nucleico , Recombinação V(D)J , Animais , Camundongos , Recombinação V(D)J/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Cromatina/metabolismoRESUMO
The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.
Assuntos
Proteínas de Choque Térmico HSP70 , Chaperonas Moleculares , Humanos , Sequência de Aminoácidos , Genômica , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , FilogeniaRESUMO
B lymphocytes develop from uncommitted precursors into immunoglobulin (antibody)-producing B cells, a major arm of adaptive immunity. Progression of early progenitors to antibody-expressing cells in the bone marrow is orchestrated by the temporal regulation of different gene programs at discrete developmental stages. A major question concerns how B cells control the accessibility of these genes to transcription factors. Research has implicated nucleosome remodeling ATPases as mediators of chromatin accessibility. Here, we describe studies of chromodomain helicase DNA-binding 4 (CHD4; also known as Mi-2ß) in early B cell development. CHD4 comprises multiple domains that function in nucleosome mobilization and histone binding. CHD4 is a key component of Nucleosome Remodeling and Deacetylase, or NuRD (Mi-2) complexes, which assemble with other proteins that mediate transcriptional repression. We review data demonstrating that CHD4 is necessary for B lineage identity: early B lineage progression, proliferation in response to interleukin-7, responses to DNA damage, and cell survival in vivo. CHD4-NuRD is also required for the Ig heavy-chain repertoire by promoting utilization of distal variable (VH ) gene segments in V(D)J recombination. In conclusion, the regulation of chromatin accessibility by CHD4 is essential for production of antibodies by B cells, which in turn mediate humoral immune responses to pathogens and disease.
Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Recombinação V(D)J , Linfócitos B/metabolismo , DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismoRESUMO
H2A variants are histones that carry out specialized nucleosome function during the eukaryote genome packaging. Most genes encoding H2A histone variants arose in the distant past, and have highly conserved domains and structures. Yet, novel H2A variants have continued to arise throughout the radiation of eukaryotes and disturbed the apparent tranquility of nucleosomes. These species-specific H2A variants contributed to the functional diversification of nucleosomes through changes in both their structure and expression patterns. In this short review, we discuss the evolutionary trajectories of these histone renegades in plants and animal genomes.
Assuntos
Histonas , Nucleossomos , Animais , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Plantas/genética , Plantas/metabolismo , Genoma , Evolução BiológicaRESUMO
BACKGROUND & AIMS: Pouchitis is the most common complication after restorative proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis. This American Gastroenterological Association (AGA) guideline is intended to support practitioners in the management of pouchitis and inflammatory pouch disorders. METHODS: A multidisciplinary panel of content experts and guideline methodologists used the Grading of Recommendations Assessment, Development and Evaluation framework to prioritize clinical questions, identify patient-centered outcomes, conduct an evidence synthesis, and develop recommendations for the prevention and treatment of pouchitis, Crohn's-like disease of the pouch, and cuffitis. RESULTS: The AGA guideline panel made 9 conditional recommendations. In patients with ulcerative colitis who have undergone ileal pouch-anal anastomosis and experience intermittent symptoms of pouchitis, the AGA suggests using antibiotics for the treatment of pouchitis. In patients who experience recurrent episodes of pouchitis that respond to antibiotics, the AGA suggests using probiotics for the prevention of recurrent pouchitis. In patients who experience recurrent pouchitis that responds to antibiotics but relapses shortly after stopping antibiotics (also known as "chronic antibiotic-dependent pouchitis"), the AGA suggests using chronic antibiotic therapy to prevent recurrent pouchitis; however, in patients who are intolerant to antibiotics or who are concerned about the risks of long-term antibiotic therapy, the AGA suggests using advanced immunosuppressive therapies (eg, biologics and/or oral small molecule drugs) approved for treatment of inflammatory bowel disease. In patients who experience recurrent pouchitis with inadequate response to antibiotics (also known as "chronic antibiotic-refractory pouchitis"), the AGA suggests using advanced immunosuppressive therapies; corticosteroids can also be considered in these patients. In patients who develop symptoms due to Crohn's-like disease of the pouch, the AGA suggests using corticosteroids and advanced immunosuppressive therapies. In patients who experience symptoms due to cuffitis, the AGA suggests using therapies that have been approved for the treatment of ulcerative colitis, starting with topical mesalamine or topical corticosteroids. The panel also proposed key implementation considerations for optimal management of pouchitis and Crohn's-like disease of the pouch and identified several knowledge gaps and areas for future research. CONCLUSIONS: This guideline provides a comprehensive, patient-centered approach to the management of patients with pouchitis and other inflammatory conditions of the pouch.
Assuntos
Colite Ulcerativa , Doença de Crohn , Pouchite , Proctocolectomia Restauradora , Humanos , Pouchite/diagnóstico , Pouchite/tratamento farmacológico , Pouchite/etiologia , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/cirurgia , Colite Ulcerativa/complicações , Proctocolectomia Restauradora/efeitos adversos , Doença de Crohn/diagnóstico , Antibacterianos/uso terapêutico , CorticosteroidesRESUMO
Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1. RNase J1 catalyzes 5'-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's "sliding endonuclease" activity. 5'-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5'-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5'-central bubble to trigger 5'-to-3'-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3'-exonucleases of B. subtilis-RNase R, PNPase, YhaM, and particularly RNase PH-are involved in 3'-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, â¼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.
Assuntos
Bacillus subtilis , RNA Bacteriano , RNA Bacteriano/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA não Traduzido/metabolismo , Ribonuclease Pancreático/metabolismo , Estabilidade de RNA/genéticaRESUMO
12,13-dihydroxy-9z-octadecenoic acid (12,13-DiHOME) is a linoleic acid diol derived from cytochrome P-450 (CYP) epoxygenase and epoxide hydrolase (EH) metabolism. 12,13-DiHOME is associated with inflammation and mitochondrial damage in the innate immune response, but how 12,13-DiHOME contributes to these effects is unclear. We hypothesized that 12,13-DiHOME enhances macrophage inflammation through effects on NOD-like receptor protein 3 (NLRP3) inflammasome activation. To test this hypothesis, we utilized human monocytic THP1 cells differentiated into macrophage-like cells with phorbol myristate acetate (PMA). 12,13-DiHOME present during lipopolysaccharide (LPS)-priming of THP1 macrophages exacerbated nigericin-induced NLRP3 inflammasome activation. Using high-resolution respirometry, we observed that priming with LPS+12,13-DiHOME altered mitochondrial respiratory function. Mitophagy, measured using mito-Keima, was also modulated by 12,13-DiHOME present during priming. These mitochondrial effects were associated with increased sensitivity to nigericin-induced mitochondrial depolarization and reactive oxygen species production in LPS+12,13-DiHOME-primed macrophages. Nigericin-induced mitochondrial damage and NLRP3 inflammasome activation in LPS+12,13-DiHOME-primed macrophages were ablated by the mitochondrial calcium uniporter (MCU) inhibitor, Ru265. 12,13-DiHOME present during LPS-priming also enhanced nigericin-induced NLRP3 inflammasome activation in primary murine bone marrow-derived macrophages. In summary, these data demonstrate a pro-inflammatory role for 12,13-DiHOME by enhancing NLRP3 inflammasome activation in macrophages.
Assuntos
Inflamassomos , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Inflamassomos/metabolismo , Animais , Humanos , Camundongos , Células THP-1 , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Ácido Linoleico/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Alzheimer's disease (AD) is currently an incurable neurodegenerative disorder and is the most common etiological cause of dementia. Consequently, it has severe burden on its patients and on their caregivers and represents a global health concern. Clinical investigations have indicated that a dysregulation of peripheral T cell immune homeostasis may be involved in the pathogenesis of AD, as well as in the early stages of AD, characterized by mild cognitive impairment (MCI). However, the characteristics and concomitant feasibility of the use of T-cell receptor (TCR) typing for disease diagnosis remains largely unknown. We employed a high-throughput sequencing and multidimensional bioinformatics analyses for the identification of TCR repertoires present in peripheral blood samples of 10 patients with amnestic MCI (aMCI), 10 patients with AD, and 10 healthy controls (HCs). Based on the characteristics of the TCR repertoires in the amount and diversity of combinations of V-J, the spectrum of immune defense, and differentially expressed genes (DEGs), single and specific TCR profiles were observed in the patient samples of aMCI and AD compared to profiles of HCs. In particular, the diversity of TCR clonotypes manifested a pattern of "decreased first and then increased" pattern during the progression from aMCI to AD, a pattern that was not observed in HC samples. Additionally, a total of 46 and 35 amino acid CDR3 sequences with consistent and reverse expressive abundance with diversity of TCR clonotypes were identified, respectively. Taken together, we provide novel and essential preliminary evidence demonstrating the presence of diversity of T cell repertoires from differentially expressed V-J gene segments and amino acid clonotypes using peripheral blood samples from patients with AD, aMCI, and from HC. Such findings have the potential to reveal potential mechanisms through which aMCI progresses to AD and provide a reference for the future development of immune-related diagnoses and therapies for AD.