RESUMEN
Transcriptional regulatory networks play a central role in optimizing cell survival. How DNA binding domains and cis-regulatory DNA binding sequences have co-evolved to allow the expansion of transcriptional networks and how this contributes to cellular fitness remains unclear. Here we experimentally explore how the complex G1/S transcriptional network evolved in the budding yeast Saccharomyces cerevisiae by examining different chimeric transcription factor (TF) complexes. Over 200 G1/S genes are regulated by either one of the two TF complexes, SBF and MBF, which bind to specific DNA binding sequences, SCB and MCB, respectively. The difference in size and complexity of the G1/S transcriptional network across yeast species makes it well suited to investigate how TF paralogs (SBF and MBF) and DNA binding sequences (SCB and MCB) co-evolved after gene duplication to rewire and expand the network of G1/S target genes. Our data suggests that whilst SBF is the likely ancestral regulatory complex, the ancestral DNA binding element is more MCB-like. G1/S network expansion took place by both cis- and trans- co-evolutionary changes in closely related but distinct regulatory sequences. Replacement of the endogenous SBF DNA-binding domain (DBD) with that from more distantly related fungi leads to a contraction of the SBF-regulated G1/S network in budding yeast, which also correlates with increased defects in cell growth, cell size, and proliferation.
Asunto(s)
Evolución Molecular , Fase G1/genética , Duplicación de Gen , Aptitud Genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Sitios de Unión , Redes Reguladoras de Genes , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Recent studies of Haloferax volcanii have begun to elucidate the steps of N-glycosylation in Archaea, where this universal post-translational modification remains poorly described. In Hfx. volcanii, a series of Agl proteins catalyzes the assembly and attachment of a N-linked pentasaccharide to the S-layer glycoprotein. Although roles have been assigned to the majority of Agl proteins, others await description. In the following, the contribution of AglR to N-glycosylation was addressed. METHODS: A combination of bioinformatics, gene deletion, mass spectrometry and metabolic radiolabeling served to show a role for AglR in archaeal N-glycosylation at both the dolichol phosphate and reporter glycoprotein levels. RESULTS: The modified behavior of the S-layer glycoprotein isolated from cells lacking AglR points to an involvement of this protein in N-glycosylation. In cells lacking AglR, glycan-charged dolichol phosphate, including mannose-charged dolichol phosphate, accumulates. At the same time, the S-layer glycoprotein does not incorporate mannose, the final subunit of the N-linked pentasaccharide decorating this protein. AglR is a homologue of Wzx proteins, annotated as flippases responsible for delivering lipid-linked O-antigen precursor oligosaccharides across the bacterial plasma membrane during lipopolysaccharide biogenesis. CONCLUSIONS: The effects resulting from aglR deletion are consistent with AglR interacting with dolichol phosphate-mannose, possibly acting as a dolichol phosphate-mannose flippase or contributing to such activity. GENERAL SIGNIFICANCE: Little is known of how lipid-linked oligosaccharides are translocated across membrane during N-glycosylation. The possibility of Hfx. volcanii AglR mediating or contributing to flippase activity could help address this situation.
Asunto(s)
Proteínas Arqueales/fisiología , Haloferax volcanii/metabolismo , Manosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Cromatografía Liquida , Glicosilación , Haloferax volcanii/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Organismos Modificados Genéticamente , Polisacáridos/química , Procesamiento Proteico-Postraduccional/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Of the many post-translational modifications proteins can undergo, glycosylation is the most prevalent and the most diverse. Today, it is clear that both N-glycosylation and O-glycosylation, once believed to be restricted to eukaryotes, also transpire in Bacteria and Archaea. Indeed, prokaryotic glycoproteins rely on a wider variety of monosaccharide constituents than do those of eukaryotes. In recent years, substantial progress in describing the enzymes involved in bacterial and archaeal glycosylation pathways has been made. It is becoming clear that enhanced knowledge of bacterial glycosylation enzymes may be of therapeutic value, while the demonstrated ability to introduce bacterial glycosylation genes into Escherichia coli represents a major step forward in glyco-engineering. A better understanding of archaeal protein glycosylation provides insight into this post-translational modification across evolution as well as protein processing under extreme conditions. Here, we discuss new structural and biosynthetic findings related to prokaryotic protein glycosylation, until recently a neglected topic.
Asunto(s)
Archaea/metabolismo , Proteínas Arqueales/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Archaea/química , Bacterias/química , Campylobacter jejuni/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilación , Haloferax volcanii/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria gonorrhoeae/metabolismo , Conformación ProteicaRESUMEN
The sensitivity of the protein-folding environment to chaperone disruption can be highly tissue-specific. Yet, the organization of the chaperone system across physiological human tissues has received little attention. Through computational analyses of large-scale tissue transcriptomes, we unveil that the chaperone system is composed of core elements that are uniformly expressed across tissues, and variable elements that are differentially expressed to fit with tissue-specific requirements. We demonstrate via a proteomic analysis that the muscle-specific signature is functional and conserved. Core chaperones are significantly more abundant across tissues and more important for cell survival than variable chaperones. Together with variable chaperones, they form tissue-specific functional networks. Analysis of human organ development and aging brain transcriptomes reveals that these functional networks are established in development and decline with age. In this work, we expand the known functional organization of de novo versus stress-inducible eukaryotic chaperones into a layered core-variable architecture in multi-cellular organisms.
Asunto(s)
Chaperonas Moleculares/metabolismo , Especificidad de Órganos , Envejecimiento/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Línea Celular , Secuencia Conservada , Evolución Molecular , Regulación de la Expresión Génica , Humanos , Ratones , Chaperonas Moleculares/genética , Sistemas de Lectura Abierta/genética , Especificidad de Órganos/genéticaRESUMEN
Like the Eukarya and Bacteria, the Archaea also perform N glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO_1517 to agl genes encoding known elements of the H. volcanii N-glycosylation process. By combining the deletion of HVO_1517 with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of aglJ, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while aglJ can be deleted, H. volcanii surface layer integrity is compromised in the absence of the encoded protein.
Asunto(s)
Proteínas Arqueales/metabolismo , Metabolismo de los Hidratos de Carbono , Regulación de la Expresión Génica Arqueal/fisiología , Haloferax volcanii/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Arqueales/genética , Proteínas Portadoras/metabolismo , Eliminación de Gen , Glicosilación , Haloferax volcanii/genética , Hexosas/metabolismo , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
Proteins in all three domains of life can experience N-glycosylation. The steps involved in the archaeal version of this post-translational modification remain largely unknown. Hence, as the next step in ongoing efforts to identify components of the N-glycosylation pathway of the halophilic archaeon Haloferax volcanii, the involvement of three additional gene products in the biosynthesis of the pentasaccharide decorating the S-layer glycoprotein was demonstrated. The genes encoding AglF, AglI and AglG are found immediately upstream of the gene encoding the archaeal oligosaccharide transferase, AglB. Evidence showing that AglF and AglI are involved in the addition of the hexuronic acid found at position three of the pentasaccharide is provided, while AglG is shown to contribute to the addition of the hexuronic acid found at position two. Given their proximities in the H. volcanii genome, the transcription profiles of aglF, aglI, aglG and aglB were considered. While only aglF and aglI share a common promoter, transcription of the four genes is co-ordinated, as revealed by determining transcript levels in H. volcanii cells raised in different growth conditions. Such changes in N-glycosylation gene transcription levels offer additional support for the adaptive role of this post-translational modification in H. volcanii.
Asunto(s)
Proteínas Arqueales/metabolismo , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Arqueales/genética , Regulación de la Expresión Génica Arqueal , Glicosilación , Glicoproteínas de Membrana/genética , Viabilidad Microbiana , Regiones Promotoras Genéticas , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
Archaea, like Eukarya and Bacteria, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea.
Asunto(s)
Proteínas Arqueales/metabolismo , Haloferax volcanii/enzimología , Manosiltransferasas/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Mapeo Cromosómico , Eliminación de Gen , Glicosilación , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Manosiltransferasas/análisis , Manosiltransferasas/genética , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Molecular co-evolution is manifested by compensatory changes in proteins designed to enable adaptation to their natural environment. In recent years, bioinformatics approaches allowed for the detection of co-evolution at the level of the whole protein or of specific residues. Such efforts enabled prediction of protein-protein interactions, functional assignments of proteins and the identification of interacting residues, thereby providing information on protein structure. Still, despite such advances, relatively little is known regarding the functional implications of sequence divergence resulting from protein co-evolution. While bioinformatics approaches usually analyze thousands of proteins to obtain a broad view of protein co-evolution, experimental evaluation of protein co-evolution serves to study only individual proteins. In this review, we describe recent advances in bioinformatics and experimental efforts aimed at examining protein co-evolution. Accordingly, we discuss possible modes of crosstalk between the bioinformatics and experimental approaches to facilitate the identification of co-evolutionary signals in proteins and to understand their implications for the structure and function of proteins.
Asunto(s)
Biología Computacional , Evolución Molecular , Proteínas/análisis , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMEN
Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.
Asunto(s)
Archaea/química , Proteínas Arqueales/química , Glicoproteínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Archaea/metabolismo , Proteínas Arqueales/metabolismo , Asparagina/metabolismo , Proteínas Bacterianas/química , Células Eucariotas/química , Glicoproteínas/metabolismo , Glicosilación , Datos de Secuencia Molecular , Análisis de Secuencia de ProteínaRESUMEN
In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.
Asunto(s)
Proteínas Arqueales/metabolismo , Glicoproteínas/metabolismo , Haloferax volcanii/metabolismo , Glicosilación , Oligosacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In this study, characterization of the N-glycosylation process in the haloarchaea Haloferax volcanii was undertaken. Initially, putative Hfx. volcanii homologues of genes involved in eukaryal or bacterial N-glycosylation were identified by bioinformatics. Reverse transcription polymerase chain reaction (RT-PCR) confirmed that the proposed N-glycosylation genes are transcribed, indicative of true proteins being encoded. Where families of related gene sequences were detected, differential transcription of family members under a variety of physiological and environmental conditions was shown. Gene deletions point to certain genes, like alg11, as being essential yet revealed that others, such as the two versions of alg5, are not. Deletion of alg5-A did, however, lead to slower growth and interfered with surface (S)-layer glycoprotein glycosylation, as detected by modified migration on SDS-PAGE and glycostaining approaches. As deletion of stt3, the only component of the oligosaccharide transferase complex detected in Archaea, did not affect cell viability, it appears that N-glycosylation is not essential in Hfx. volcanii. Deletion of stt3 did, nonetheless, hinder both cell growth and S-layer glycoprotein glycosylation. Thus, with genes putatively involved in Hfx. volcanii protein glycosylation identified and the ability to address the roles played by the encoded polypeptides in modifying a reporter glycoprotein, the steps of the archaeal N-glycosylation pathway can be defined.
Asunto(s)
Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Haloferax volcanii/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Arqueales/genética , Eliminación de Gen , Regulación de la Expresión Génica Arqueal , Glicosilación , Haloferax volcanii/crecimiento & desarrollo , Haloferax volcanii/metabolismo , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Lípidos/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Sistemas de Lectura Abierta , Polisacáridos/química , Polisacáridos/metabolismo , Subunidades de ProteínaRESUMEN
The teleost non-specific cytotoxic cells (NCC) are evolutionary precursors of the mammalian natural killer (NK) cells and an important element of innate immunity. The non-specific cytotoxic cell receptor protein (NCCRP1) is a characteristic cell surface protein with main functions in target cell recognition and cytotoxicity with sequence information available for many species of fish. We have isolated a cDNA encoding the Axolotl homologue of fish NCCRP1 out of limb regeneration blastema and analysed its expression by RT-PCR. Sequence analysis revealed a high degree of homology with teleost NCCRP1 on nucleotide and deduced amino acid levels. NCCRP1 contains a conserved C-terminal F-box-associated domain (FBA) and proline-rich motifs (PRM) characteristic for this protein family. NCCRP1 is expressed in multiple tissues with high levels in limb regeneration blastema. The present work describes for the first time the cloning of the NCCRP1 gene in a tetrapod vertebrate providing a valuable link between fish and higher vertebrates. Our findings suggest the existence of NCC in axolotl and a role of the innate immune system in the processes of limb regeneration.