RESUMEN
While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.
Asunto(s)
Oxidorreductasas de Alcohol , Polimerizacion , Neoplasias de la Próstata , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/química , Microscopía por Crioelectrón , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Unión Proteica , Células HEK293 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/química , Secuencias de Aminoácidos , Proteínas Co-Represoras/metabolismo , Proteínas Co-Represoras/genéticaRESUMEN
Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.
Asunto(s)
Melanoma , Proteínas de Dominio T Box , Expresión Génica , Humanos , Melanoma/genética , Melanoma/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Retinoic acid-induced protein 2 is a human protein of 530 residues encoded by the RAI2 gene (Q9Y5P3; RAI2_HUMAN). RAI2 is a novel tumor suppressor protein whose depletion in breast cancer cell lines results in the downregulation of several genes associated with differentiation along with increased invasiveness and aggressive tumor phenotype of the cells. The role of the protein is specified to be a transcriptional regulator that promotes chromosomal stability and hence controls the expression of several regulators of cancer and metastasis. Structurally, RAI2 remains an unknown entity and, hence, to obtain a detailed view on the structure function relationship we report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of the C-terminal region (a.a. 303-451 of UniProt Q9Y5P3) of RAI2.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/química , Espectroscopía de Protones por Resonancia Magnética , Algoritmos , Humanos , Isótopos de NitrógenoRESUMEN
Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
Asunto(s)
Hemo/metabolismo , Interleucina-1/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Citocinas/agonistas , Citocinas/química , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Mediadores de Inflamación/agonistas , Mediadores de Inflamación/química , Mediadores de Inflamación/metabolismo , Interleucina-1/agonistas , Interleucina-1/química , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Psoriasis/metabolismo , Psoriasis/patología , Relación Estructura-Actividad , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Cystathionine-ß-synthase (CBS) belongs to a large family of pyridoxal 5'-phosphate (PLP)-dependent enzymes, responsible for the sulfur metabolism. The heme-dependent protein CBS is part of regulatory pathways also involving the gasotransmitter hydrogen sulfide. Malfunction of CBS can lead to pathologic conditions like cancer, cardiovascular and neurodegenerative disorders. Truncation of residues 1-40, absent in X-ray structures of CBS, reduces but does not abolish the activity of the enzyme. Here we report the NMR resonance assignment and heme interaction studies for the N-terminal peptide stretch of CBS. We present NMR-spectral evidence that residues 1-40 constitute an intrinsically disordered region in CBS and interact with heme via a cysteine-proline based motif.
Asunto(s)
Cistationina betasintasa/química , Hemo/química , Proteínas Intrínsecamente Desordenadas/química , Péptidos/química , Fosfato de Piridoxal/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hemo/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Cinética , Resonancia Magnética Nuclear Biomolecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TermodinámicaRESUMEN
Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the (1)H, (13)C, and (15)N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.
Asunto(s)
Interleucina-1/química , Interleucina-1/metabolismo , Resonancia Magnética Nuclear Biomolecular , Inflamación/metabolismoRESUMEN
BACKGROUND: The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS: To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS: We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 µM. CONCLUSIONS: It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE: Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Hemo/farmacología , Secuencias de Aminoácidos , Cloranfenicol O-Acetiltransferasa/química , Espectroscopía de Resonancia por Spin del Electrón , Histidina , Espectroscopía de Resonancia Magnética , Espectrometría Raman , TirosinaRESUMEN
The importance of heme as a transient regulatory molecule has become a major focus in biochemical research. However, detailed information about the molecular basis of transient heme-protein interactions is still missing. We report an in-depth structural analysis of Fe(III) heme-peptide complexes by a combination of UV/Vis, resonance Raman, and 2D-NMR spectroscopic methods. The experiments reveal insights both into the coordination to the central iron ion and into the spatial arrangement of the amino acid sequences interacting with protoporphyrin IX. Cysteine-based peptides display different heme-binding behavior as a result of the existence of ordered, partially ordered, and disordered conformations in the heme-unbound state. Thus, the heme-binding mode is clearly the consequence of the nature and flexibility of the residues surrounding the iron ion coordinating cysteine. Our analysis reveals scenarios for transient binding of heme to heme-regulatory motifs in proteins and demonstrates that a thorough structural analysis is required to unravel how heme alters the structure and function of a particular protein.
Asunto(s)
Cisteína/metabolismo , Compuestos Férricos/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Protoporfirinas/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Cisteína/química , Compuestos Férricos/química , Estructura Terciaria de Proteína , Protoporfirinas/químicaRESUMEN
A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of (1) J CαN and (2) J CαN couplings to transfer the (15)N x magnetisation from amino acid residue 'i' to adjacent residues via the application of a band-selective (15)N-(13)C(α) heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [(15) N,(13) C']- and [(13) C',(1) H(N) ]-correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H(N) and H(α) correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side-chain (1) H and (13) C assignments, employing sequential (1) H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side-chain chemical shifts is demonstrated experimentally for the 61-residue [(13) C,(15) N]-labelled peptide of a voltage-gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Estándares de ReferenciaRESUMEN
Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of â¼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.
Asunto(s)
Hemo , Canal de Potasio Kv1.4 , Animales , Cristalografía por Rayos X , Hemo/química , Hemo/genética , Hemo/metabolismo , Transporte Iónico/fisiología , Canal de Potasio Kv1.4/química , Canal de Potasio Kv1.4/genética , Canal de Potasio Kv1.4/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Xenopus laevisRESUMEN
Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420-430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420-430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV-vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CP-peptide-heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzyme's activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.