RESUMEN

Organisms possess a wide variety of proteins with diverse amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various problematic nascent peptide sequences within the exit tunnel. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that ATP hydrolysis-coupled structural rearrangement and the interdomain linker sequence are pivotal for handling 'hard-to-translate' nascent peptides. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli , Péptidos , Secuencia de Aminoácidos , Transportadoras de Casetes de Unión a ATP/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Péptidos/metabolismo , Péptidos/química , Péptidos/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Ribosomas/genética

RESUMEN

Therapeutic agents targeting B cells, such as rituximab, have been proven to be effective in several diseases. However, since this therapy targets the whole B cell population, there are still concerns about critical adverse events. Therefore, a new type of antibody that can specifically deplete a particular type of B cell may have the potential to become an efficient and safer therapy. Membrane-bound IgA1 (mIgA1) is expressed on soluble IgA1 (sIgA1) producing B cells and/or their precursor, B cells. Although most of the amino acid sequence in the N-terminal regions of the extracellular domains of mIgA1 and sIgA1 is shared, there is a membrane type-specific region, named the membrane-bound immunoglobulin isotype-specific (migis-α) region, at the membrane-proximal region of mIgA1. We hypothesized that the migis-α region would be a suitable antigen for therapeutic antibodies to target mIgA1-expressing B cells without binding to sIgA1, which may cause undesired adverse effects and poor pharmacokinetics (PK). We established two anti-migis-α monoclonal antibodies (mAbs), KM4641 and KM4644, by immunization of the synthetic peptide corresponding to an migis-α region. These mAbs both showed robust binding to mIgA1-expressing transfectant cells. As we expected, neither mAbs bound to sIgA1 and we found that the mAbs recognized different seven to eight amino acid sequences within the migis-α region. Furthermore, the rat-human chimeric type of both mAbs showed antibody-dependent cellular cytotoxicity against mIgA1-expressing transfectant cells. Taken together, this study showed that established mAbs have therapeutic potential in IgA-related diseases, such as IgA nephropathy.

Asunto(s)

Anticuerpos Monoclonales , Glomerulonefritis por IGA , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Inmunoglobulina A , Ratas , Receptores de Antígenos de Linfocitos B

RESUMEN

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

RESUMEN

Cytochrome P450 1A1 (CYP1A1) catalyzes the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and is transcriptionally regulated by the aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex upon exposure to PAHs. Accordingly, inhibition of CYP1A1 expression reduces production of carcinogens from PAHs. Although transcription of the CYP1A1 gene is known to be repressed by transforming growth factor-ß (TGF-ß), how TGF-ß signaling is involved in the suppression of CYP1A1 gene expression has yet to be clarified. In this study, using mammalian cell lines, along with shRNA-mediated gene silencing, CRISPR/Cas9-based genome editing, and reporter gene and quantitative RT-PCR assays, we found that TGF-ß signaling dissociates the B[a]P-mediated AhR/ARNT heteromeric complex. Among the examined Smads, Smad family member 3 (Smad3) strongly interacted with both AhR and ARNT via its MH2 domain. Moreover, hypoxia-inducible factor 1α (HIF-1α), which is stabilized upon TGF-ß stimulation, also inhibited AhR/ARNT complex formation in the presence of B[a]P. Thus, TGF-ß signaling negatively regulated the transcription of the CYP1A1 gene in at least two different ways. Of note, TGF-ß abrogated DNA damage in B[a]P-exposed cells. We therefore conclude that TGF-ß may protect cells against carcinogenesis because it inhibits CYP1A1-mediated metabolic activation of PAHs as part of its anti-tumorigenic activities.

Asunto(s)

Citocromo P-450 CYP1A1/genética , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células A549 , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Benzo(a)pireno/toxicidad , Células COS , Chlorocebus aethiops , Citocromo P-450 CYP1A1/metabolismo , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Pirenos , Transducción de Señal , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

Galactose-deficient IgA1 (Gd-IgA1) that exposes GalNAc or sialylated GalNAc has been shown to be associated with disease activity of IgA nephropathy (IgAN). In a previous report, we established an enzyme-linked immunosorbent assay that measures human Gd-IgA1 using a specific monoclonal antibody KM55 (KM55 mAb), and showed that patients with IgAN contain a higher level of serum Gd-IgA1 than other types of renal diseases. Recently, we also found that the KM55 mAb specifically recognized the glomerular-deposited Gd-IgA1 in renal biopsy. In this study, we aimed to analyze the epitope of KM55 mAb using synthesized peptides corresponding to the hinge region of IgA1 with GalNAc moiety on putative glycosylated Ser/Thr residues, which are Thr225, Thr228, Ser230, Ser232, and Thr236. Binding analysis to single GalNAc-modified hinge region peptide of IgA1 showed that Thr225 with GalNAc is required for recognition of KM55. PST(GalNAC)PP motif was required for KM55 mAb to recognize hinge region peptide of IgA1 which is shown by binding assay with deletion peptide. This result was confirmed by binding of KM55 mAb against peptide with GalNAc at Thr233, which resulted in containing another PST(GalNAC)PP motif. Taken together, we concluded that the epitope of Gd-IgA1-specific KM55 mAb is PST(GalNAc)PP motif.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Galactosa/deficiencia , Galactosa/inmunología , Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/química , Inmunoglobulina A/inmunología , Especificidad de Anticuerpos , Glicosilación , Humanos

RESUMEN

Galactose-deficient IgA1 has been proposed as an important effector molecule in IgA nephropathy (IgAN). We previously showed that the galactose-deficient IgA1-specific monoclonal antibody KM55 can detect circulating galactose-deficient IgA1 in patients with IgAN, enabling us to study the molecular roles of galactose-deficient IgA1. Herein, we further examined the pathophysiological significance of galactose-deficient IgA1 in glomerular deposits of patients with IgAN by immunohistochemistry using KM55. Immunostaining of galactose-deficient IgA1 with KM55 was performed in paraffin-embedded sections of renal biopsy specimens from 48 patients with IgAN and 49 patients with other renal diseases such as lupus nephritis, HCV-related nephropathy, IgA vasculitis with nephritis (IgA-VN), and membranous nephropathy. Glomerular galactose-deficient IgA1 was specifically detected in IgAN and IgA-VN but not in the other renal diseases. Galactose-deficient IgA1 was localized predominantly in the mesangial region as IgA deposition. However, galactose-deficient IgA1 was not detected in patients with lupus nephritis accompanied by glomerular IgA deposition. Thus, our study strongly suggests that IgAN and IgA-VN have a shared feature regarding galactose-deficient IgA1-oriented pathogenesis.

Asunto(s)

Galactosa/deficiencia , Glomerulonefritis por IGA/sangre , Inmunoglobulina A/inmunología , Glomérulos Renales/química , Vasculitis/inmunología , Anticuerpos Monoclonales/inmunología , Biopsia , Técnica del Anticuerpo Fluorescente , Galactosa/sangre , Glomerulonefritis por IGA/diagnóstico , Glomerulonefritis por IGA/inmunología , Humanos , Inmunoglobulina A/sangre , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Microscopía Fluorescente , Vasculitis/sangre , Vasculitis/diagnóstico

RESUMEN

Liver X receptor (LXR)α and LXRß belong to the nuclear receptor superfamily and play central roles in the transcriptional control of lipid metabolism. We describe a novel LXR target, midline-1-interacting G12-like protein (MIG12), which has been recently identified as an acetyl-coenzyme A carboxylase-binding protein. The binding causes the induction of de novo fatty acid (FA) synthesis through the activation of acetyl-coenzyme A carboxylase (a rate-limiting enzyme for de novo FA synthesis). Luciferase reporter gene assays using the MIG12 gene promoter revealed the existence of a LXR-responsive element (LXRE) and carbohydrate-responsive element-binding protein (ChREBP)-responsive element named LXRE3 and carbohydrate response element 1, respectively. Deletion and mutation of LXRE3 and carbohydrate response element 1 abolished LXR and ChREBP responsiveness, respectively. Electrophoretic mobility shift assays demonstrated that the LXRα/retinoid X receptor α complex was bound to LXRE3. Treatment with high glucose concentration, which leads ChREBP activation, or LXR activator stimulated MIG12 expression in rat primary hepatocytes, and combined treatment further stimulated MIG12 expression. Furthermore, hepatic expression of MIG12 in mice was induced by refeeding. Overexpression of MIG12 stimulated and knockdown of MIG12 attenuated LXR ligand-stimulated de novo FA synthesis and triacylglycerol accumulation. These results indicate that MIG12 is a mediator for stimulation of lipogenesis by LXR activation in the liver.

Asunto(s)

Lipogénesis/genética , Luciferasas/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Animales , Anticolesterolemiantes/farmacología , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células Cultivadas , Ácidos Grasos/biosíntesis , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Hepatocitos/metabolismo , Humanos , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado , Luciferasas/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Nucleares Huérfanos/genética , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Triglicéridos/metabolismo