RESUMEN
Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3' UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3' UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3' UTR.
Asunto(s)
Codón sin Sentido/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/genética , Factores de Terminación de Péptidos/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismo , Northern Blotting , Citoplasma/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Factores de Terminación de Péptidos/genética , Poli A/metabolismo , Proteína I de Unión a Poli(A)/genética , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , Ribosomas/metabolismoRESUMEN
Signal recognition particle (SRP)-dependent protein targeting is a universally conserved process that delivers proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum membrane in eukaryotes. Crucial during targeting is the transfer of the ribosome-nascent chain complex (RNC) from SRP to the Sec translocon. In eukaryotes, this step is co-ordinated by the SRß subunit of the SRP receptor (SR), which probably senses a vacant translocon by direct interaction with the translocon. Bacteria lack the SRß subunit and how they co-ordinate RNC transfer is unknown. By site-directed cross-linking and fluorescence resonance energy transfer (FRET) analyses, we show that FtsY, the bacterial SRα homologue, binds to the exposed C4/C5 loops of SecY, the central component of the bacterial Sec translocon. The same loops serve also as binding sites for SecA and the ribosome. The FtsY-SecY interaction involves at least the A domain of FtsY, which attributes an important function to this so far ill-defined domain. Binding of FtsY to SecY residues, which are also used by SecA and the ribosome, probably allows FtsY to sense an available translocon and to align the incoming SRP-RNC with the protein conducting channel. Thus, the Escherichia coli FtsY encompasses the functions of both the eukaryotic SRα and SRß subunits in one single protein.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Proteínas de Transporte de Membrana/química , Receptores Citoplasmáticos y Nucleares/química , Receptores de Péptidos/química , Ribosomas/química , Partícula de Reconocimiento de Señal/química , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Espectrometría de Masas , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Ribosomas/metabolismo , Canales de Translocación SEC , Proteína SecA , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
The growing need for biologics to be administered subcutaneously and ocularly, coupled with certain indications requiring high doses, has resulted in an increase in drug substance (DS) and drug product (DP) protein concentrations. With this increase, more emphasis must be placed on identifying critical physico-chemical liabilities during drug development, including protein aggregation, precipitation, opalescence, particle formation, and high viscosity. Depending on the molecule, liabilities, and administration route, different formulation strategies can be used to overcome these challenges. However, due to the high material requirements, identifying optimal conditions can be slow, costly, and often prevent therapeutics from moving rapidly into the clinic/market. In order to accelerate and derisk development, new experimental and in-silico methods have emerged that can predict high concentration liabilities. Here, we review the challenges in developing high concentration formulations, the advances that have been made in establishing low mass and high-throughput predictive analytics, and advances in in-silico tools and algorithms aimed at identifying risks and understanding high concentration protein behavior.
Asunto(s)
Desarrollo de Medicamentos , Preparaciones Farmacéuticas/química , Desarrollo de Medicamentos/métodos , ViscosidadRESUMEN
Anticalin® proteins have been proven as versatile clinical stage biotherapeutics. Due to their small size (â¼20 kDa), they harbor a short intrinsic plasma half-life which can be extended, e.g., by fusion with IgG or Fc. However, for antagonism of co-immunostimulatory Tumor Necrosis Factor Receptor Superfamily (TNFRSF) members in therapy of autoimmune and inflammatory diseases, a monovalent, pharmacokinetically optimized Anticalin protein format that avoids receptor clustering and therefore potential activation is favored. We investigated the suitability of an affinity-improved streptococcal Albumin-Binding Domain (ABD) and the engineered Fab-selective Immunoglobulin-Binding Domain (IgBD) SpGC3Fab for plasma Half-Life Extension (HLE) of an OX40-specific Anticalin and bispecific Duocalin proteins, neutralizing OX40 and a second co-immunostimulatory TNFRSF member. The higher affinity of ABD fusion proteins to human serum albumin (HSA) and Mouse Serum Albumin (MSA), with a 4 to 5-order of magnitude lower KD compared with the binding affinity of IgBD fusions to human/mouse IgG, translated into longer terminal plasma half-lives (t 1/2). Hence, the anti-OX40 Anticalin-ABD protein reached t 1/2 values of â¼40 h in wild-type mice and 110 h in hSA/hFcRn double humanized mice, in contrast to â¼7 h observed for anti-OX40 Anticalin-IgBD in wild-type mice. The pharmacokinetics of an anti-OX40 Anticalin-Fc fusion protein was the longest in both models (t 1/2 of 130 h and 146 h, respectively). Protein formats composed of two ABDs or IgBDs instead of one single HLE domain clearly showed longer presence in the circulation. Importantly, Anticalin-ABD and -IgBD fusions showed OX40 receptor binding and functional competition with OX40L-induced cellular reactivity in the presence of albumin or IgG, respectively. Our results suggest that fusion to ABD or IgBD can be a versatile platform to tune the plasma half-life of Anticalin proteins in response to therapeutic needs.
RESUMEN
BACKGROUND: The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRbeta subunit is an integral membrane protein, which tethers the SRP-interacting SRalpha subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRbeta subunit and consists of only the SRalpha homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting. RESULTS: In the current study we have determined the contribution of soluble FtsY to co-translational targeting in vitro and have re-analysed the localization of FtsY in vivo by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs) in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in E. coli. In agreement with this observation, our in vivo analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species in vivo. CONCLUSION: The exact function of the SRP receptor (SR) in bacteria has so far been enigmatic. Our data show that the bacterial SR is almost exclusively membrane-bound in vivo, indicating that the presence of a soluble SR is probably an artefact of cell fractionation. Thus, co-translational targeting in bacteria does not involve the formation of a soluble SR-signal recognition particle (SRP)-ribosome nascent chain (RNC) intermediate but requires membrane contact of FtsY for efficient SRP-RNC recruitment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Retículo Endoplásmico/metabolismo , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Microscopía Fluorescente/métodos , Unión Proteica , Señales de Clasificación de Proteína , Transporte de Proteínas , Ribosomas/metabolismo , Canales de Translocación SECRESUMEN
The signal recognition particle (SRP)-dependent cotranslational targeting of proteins to the cytoplasmic membrane in bacteria or the endoplasmic reticulum membrane in eukaryotes is an essential process in most living organisms. Eukaryotic cells have been shown to respond to an impairment of the SRP pathway by (i) repressing ribosome biogenesis, resulting in decreased protein synthesis, and (ii) by increasing the expression of protein quality control mechanisms, such as chaperones and proteases. In the current study, we have analyzed how bacteria like Escherichia coli respond to a gradual depletion of FtsY, the bacterial SRP receptor. Our analyses using cell-free transcription/translation systems showed that FtsY depletion inhibits the translation of both SRP-dependent and SRP-independent proteins. This synthesis defect is the result of a multifaceted response that includes the upregulation of the ribosome-inactivating protein ribosome modulation factor (RMF). Although the consequences of these responses in E. coli are very similar to some of the effects also observed in eukaryotic cells, one striking difference is that E. coli obviously does not reduce the rate of protein synthesis by downregulating ribosome biogenesis. Instead, the upregulation of RMF leads to a direct and reversible inhibition of translation.
Asunto(s)
Proteínas Bacterianas/fisiología , Escherichia coli/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/fisiología , Ribosomas/genéticaAsunto(s)
Aptámeros de Nucleótidos/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Aptámeros de Nucleótidos/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Mediciones Luminiscentes , Proteínas de Unión al GTP Monoméricas/química , Estructura Terciaria de Proteína , ARN/metabolismo , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
We established a homogeneous luminescent oxygen channeling sensor for measuring activation states of small GTPases. The assay quantifies activated GTPases in cell lysates, can be applied to different GTPases, and can be used for multiplex screening. The study will provide guidelines for determining activation states of diverse GTPases in various biological contexts.
Asunto(s)
Técnicas Biosensibles/métodos , Mediciones Luminiscentes , Proteínas de Unión al GTP Monoméricas/metabolismo , Oxígeno/metabolismo , Animales , Activación Enzimática , Ratones , Células 3T3 NIH , Factores de TiempoRESUMEN
Evolutionary retention of duplicated genes encoding transcription-associated proteins (TAPs, comprising transcription factors and other transcriptional regulators) has been hypothesized to be positively correlated with increasing morphological complexity and paleopolyploidizations, especially within the plant kingdom. Here, we present the most comprehensive set of classification rules for TAPs and its application for genome-wide analyses of plants and algae. Using a dated species tree and phylogenetic comparative (PC) analyses, we define the timeline of TAP loss, gain, and expansion among Viridiplantae and find that two major bursts of gain/expansion occurred, coinciding with the water-to-land transition and the radiation of flowering plants. For the first time, we provide PC proof for the long-standing hypothesis that TAPs are major driving forces behind the evolution of morphological complexity, the latter in Plantae being shaped significantly by polyploidization and subsequent biased paleolog retention. Principal component analysis incorporating the number of TAPs per genome provides an alternate and significant proxy for complexity, ideally suited for PC genomics. Our work lays the ground for further interrogation of the shaping of gene regulatory networks underlying the evolution of organism complexity.
Asunto(s)
Plantas/genética , Evolución Molecular , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Estudio de Asociación del Genoma Completo , Cadenas de Markov , MicroARNs/genética , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , ARN de Planta/genética , Factores de Tiempo , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Different from eukaryotes, the bacterial signal recognition particle (SRP) receptor lacks a membrane-tethering SRP receptor (SR) beta subunit and is composed of only the SR alpha homologue FtsY. FtsY is a modular protein composed of three domains. The N- and G-domains of FtsY are highly similar to the corresponding domains of Ffh/SRP54 and SR alpha and constitute the essential core of FtsY. In contrast, the weakly conserved N-terminal A-domain does not seem to be essential, and its exact function is unknown. Our data show that a 14-amino-acid-long positively charged region at the N-terminus of the A-domain is involved in stabilizing the FtsY-SecYEG interaction. Mutant analyses reveal that the positively charged residues are crucial for this function, and we propose that the 14-amino-acid region serves as a transient lipid anchor. In its absence, the activity of FtsY to support cotranslational integration is reduced to about 50%. Strikingly, in vivo, a truncated isoform of FtsY that lacks exactly these first 14 amino acids exists. Different from full-length FtsY, which primarily cofractionates with the membrane, the N-terminally truncated isoform is primarily present in the soluble fraction. Mutating the conserved glycine residue at position 14 prevents the formation of the truncated isoform and impairs the activity of FtsY in cotranslational targeting. These data suggest that membrane binding and function of FtsY are in part regulated by proteolytic cleavage of the conserved 14-amino-acid motif.