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Alberto J L Macario

Publications and source records attributed to Alberto J L Macario.

17 recordsLinked to original sources
PubMed2007

Molecular chaperones: multiple functions, pathologies, and potential applications.

Cell stressors are ubiquitous and frequent, challenging cells often, which leads to the stress response with activation of anti-stress mechanisms. These mechanisms involve a variety of molecules, including molecular chaperones also known as heat-shock proteins (Hsp). The chaperones treated in this article are proteins that assist other proteins to fold, refold, travel to their place of residence (cytosol, organelle, membrane, extracellular space), and translocate across membranes. Molecular chaperones participate in a variety of physiological processes and are widespread in organisms, tissues, and cells. It follows that chaperone failure will have an impact, possibly serious, on one or more cellular function, which may lead to disease. Chaperones must recognize and interact with proteins in need of assistance or client polypeptides (e.g., nascent at the ribosome, or partially denatured by stressors), and have to interact with other chaperones because the chaperoning mechanism involves teams of chaperone molecules, i.e., multimolecular assemblies or chaperone machines. Consequently, chaperone molecules have structural domains with distinctive functions: bind the client polypeptide, interact with other chaperone molecules to build a machine, and interact with other complexes that integrate the chaperoning network. Also, various chaperones have ATP-binding and ATPase sites because the chaperoning process requires as, a rule, energy from ATP hydrolysis. Alterations in any one of these domains due to a mutation or an aberrant post-translational modification can disrupt the chaperoning process and cause diseases termed chaperonopathies. This article presents the pathologic concept of chaperonopathy with examples, and discusses the potential of using chaperones (genes or proteins) in treatment (chaperonotherapy). In addition, emerging topics within the field of study of chaperones (chaperonology) are highlighted, e.g., genomics (chaperonomics), systems biology, extracellular chaperones, and anti-chaperone antibodies.

Aging↗
PubMed2006

Chaperonomics, a new tool to study ageing and associated diseases.

The participation of molecular chaperones in the process of senescence and in the mechanisms of age-related diseases is currently under investigation in many laboratories. However, accurate, complete information about the number and diversity of chaperone genes in any given genome is scarce. Consequently, the results of efforts aimed at elucidating the role of chaperones in ageing and disease are often confusing and contradictory. To remedy this situation, we have developed chaperonomics, including means to identify and characterize chaperone genes and their families applicable to humans and model organisms. The problem is difficult because in eukaryotic organisms chaperones have evolved into complex multi-gene families. For instance, the occurrence of multiple paralogs in a single genome makes it difficult to interpret results if consideration is not given to the fact that similar but distinct chaperone genes can be differentially expressed in separate cellular compartments, tissues, and developmental stages. The availability of complete genome sequences allows implementation of chaperonomics with the purpose of understanding the composition of chaperone families in all cell compartments, their evolutionary and functional relations and, ultimately, their role in pathogenesis. Here, we present a series of concatenated, complementary procedures for identifying, characterizing, and classifying chaperone genes in genomes and for elucidating evolutionary relations and structural features useful in predicting functional properties. We illustrate the procedures with applications to the complex family of hsp70 genes and show that the kind of data obtained can provide a solid basis for future research.

Aging↗
PubMed2006

Evolution of a protein-folding machine: genomic and evolutionary analyses reveal three lineages of the archaeal hsp70(dnaK) gene.

The stress chaperone protein Hsp70 (DnaK) (abbreviated DnaK) and its co-chaperones Hsp40(DnaJ) (or DnaJ) and GrpE are universal in bacteria and eukaryotes but occur only in some archaea clustered in the order 5'-grpE-dnaK-dnaJ-3' in a locus termed Locus I. Three structural varieties of Locus I, termed Types I, II, and III, were identified, respectively, in Methanosarcinales, in Thermoplasmatales and Methanothermobacter thermoautotrophicus, and in Halobacteriales. These Locus I types corresponded to three groups identified by phylogenetic trees of archaeal DnaK proteins including the same archaeal subdivisions. These archaeal DnaK groups were not significantly interrelated, clustering instead with DnaKs from three bacterial lineages, Methanosarcinales with Firmicutes, Thermoplasmatales and M. thermoautotrophicus with Thermotoga, and Halobacteriales with Actinobacteria, suggesting that the three archaeal types of Locus I were acquired by independent events of lateral gene transfer. These associations, however, lacked strong bootstrap support and were sensitive to dataset choice and tree-reconstruction method. Structural features of dnaK loci in bacteria revealed that Methanosarcinales and Firmicutes shared a similar structure, also common to most other bacterial groups. Structural differences were observed instead in Thermotoga compared to Thermoplasmatales and M. thermoautotrophicus, and in Actinobacteria compared to Halobacteriales. It was also found that the association between the DnaK sequences from Halobacteriales and Actinobacteria likely reflects common biases in their amino acid compositions. Although the loci structural features and the DnaK trees suggested the possibility of lateral gene transfer between Firmicutes and Methanosarcinales, the similarity between the archaeal and the ancestral bacterial loci favors the more parsimonious hypothesis that all archaeal sequences originated from a unique prokaryotic ancestor.

Amino Acid Sequence↗
PubMed2005

Novel chaperonins in a prokaryote.

Group II chaperonins belong to the Hsp60 family occurring in archaea and eukaryotes. The archaeal chaperonins build the thermosome, which is similar to the eukaryotic CCT (chaperonin-containing TCP-1). Eukaryotes have eight subunits, and up until now, it was thought that archaea had between one and three subunits, depending on the species. We now report two novel subunits, termed Hsp60-4 and Hsp60-5, in the archaeon Methanosarcina acetivorans, which also has Hsp60-1, Hsp60-2, and Hsp60-3 with orthologs in Methanosarcinae. Hsp60-4 and Hsp60-5 occur only in M. acetivorans, which makes this organism unique in that it has the highest number of chaperonin subunits ever described for an archaeon. Evolutionary analysis suggests that either Hsp60-4 or Hsp60-5 paralogs have arisen by gene duplication with vastly increased accepted substitution rates or that they represent ancestral types found only in this species.

Amino Acid Sequence↗
PubMed2005

Genetic disorders involving molecular-chaperone genes: a perspective.

Molecular chaperones are important for maintaining a functional set of proteins in all cellular compartments. Together with protein degradation machineries (e.g., the ubiquitin-proteasome system), chaperones form the core of the cellular protein-quality control mechanism. Chaperones are proteins, and as such, they can be affected by mutations. At least 15 disorders have been identified that are associated with mutations in genes encoding chaperones, or molecules with features suggesting that they function as chaperones. These chaperonopathies and a few other candidates are presented in this article. In most cases, the mechanisms by which the defective genes contribute to the observed phenotypes are still uncharacterized. However, the reported observations definitely point to the possibility that abnormal chaperones participate in pathogenesis. The available data open novel perspectives and should encourage searches for new genetic chaperonopathies, as well as further analyses of the disorders discussed in this article, including detection of new cases.

Animals↗
PubMed2004

Evolution of assisted protein folding: the distribution of the main chaperoning systems within the phylogenetic domain archaea.

Newly made proteins must achieve a functional shape, the native configuration, before they can play their physiological roles in the cell. Proteins must also travel to the locale (e.g., the mitochondrion) in the cell where their functions are required. In these processes of folding into the native configuration and translocation to the place of work, proteins may be assisted by molecules called molecular chaperones. Stressors can unfold (denature) proteins, and genetic defects can cause misfolding and, in addition, both abnormalities can lead to polypeptide aggregation. Chaperones play a role in assisting refolding of partially denatured or misfolded proteins, thus preventing aggregation. Clearly, molecular chaperones are key cell components under normal, physiological circumstances, as well as in potentially harmful situations resulting from environmental or inherited factors. Hence, molecular chaperones constitute attractive targets for a variety of efforts aiming at improving the cell's performance, particularly under stress, to prevent disease, or at least to slow down its progression and to contain the deleterious effects of stress. In our efforts in this direction, we have undertaken to investigate the chaperoning systems of cells belonging to the phylogenetic domain Archaea. The findings reported here pertain to the distribution of the molecular chaperone machine, the chaperonins, and the prefoldins, among archaea. The genes hsp70(dnaK), hsp40(dnaJ), and grpE encoding the components of the molecular chaperone machine were present only in some archeaeal species: this contrasts with bacteria and eucarya, which do have the genes with no known exception. The group I, or bacterial, chaperonin-genes groEL and groES occured in the genomes of Methanosarcina species but were not found in any of the other archaea whose genomes have been sequenced. While all the archaea studied had between one and three chaperonins of group II (thermosome subunits), Methanosarcina acetivorans was exceptional since it had five of these chaperonins. This is the largest number of group II chaperonins ever found in a prokaryote. Furthermore, two of the M. acetivorans chaperonins were different from, albeit related to, the other known archaeal and eucaryal chaperonins of group II. Prefoldins were found in all archaea examined. Overall, the results provide clues to the evolution of the chaperoning systems, which must have played a critical role in survival since life started. Also, the data suggest new avenues of research for elucidating the evolution of assisted protein folding and for uncovering roles and interactions not yet described for these molecules.

Archaea↗
PubMed2004

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli.

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.

Adenosine Triphosphatases↗
PubMed2004

The pathology of cellular anti-stress mechanisms: a new frontier.

Exposure to stressors is an omnipresent variable for all living organisms, which have evolved anti-stress mechanisms to deal with the consequences of stress. The chaperoning systems are among these mechanisms, and their central components are the molecular chaperones that play important roles in protein biogenesis. Recent data suggest that failure of the chaperoning systems due to defective chaperones, for example, leads to pathology. Consequently, medical researchers and practitioners must now also consider the chaperoning systems, both as potentially major players in pathogenesis and as diagnostic-prognostic indicators.

Animals↗
PubMed2003

Molecular biology of stress genes in methanogens: potential for bioreactor technology.

Many agents of physical, chemical, or biological nature, have the potential for causing cell stress. These agents are called stressors and their effects on cells are due to protein denaturation. Cells, microbes, for instance, perform their physiological functions and survive stress only if they have their proteins in the necessary concentrations and shapes. To be functional a protein shape must conform to a specific three-dimensional arrangement, named the native configuration. When a stressor (e.g., temperature elevation or heat shock, decrease in pH, hypersalinity, heavy metals) hits a microbe, it causes proteins to lose their native configuration, which is to say that stressors cause protein denaturation. The cell mounts an anti-stress response: house-keeping genes are down-regulated and stress genes are activated. Among the latter are the genes that produce the Hsp70(DnaK), Hsp60, and small heat protein (sHsp) families of stress proteins. Hsp70(DnaK) is part of the molecular chaperone machine together with Hsp40(DnaJ) and GrpE, and Hsp60 is a component of the chaperonin complex. Both the chaperone machine and the chaperonins play a crucial role in assisting microbial proteins to reach their native, functional configuration and to regain it when it is partially lost due to stress. Proteins that are denatured beyond repair are degraded by proteases so they do not accumulate and become a burden to the cell. All Archaea studied to date possess chaperonins but only some methanogens have the chaperone machine. A recent genome survey indicates that Archaea do not harbor well conserved equivalents of the co-chaperones trigger factor, Hip, Hop, BAG-1, and NAC, although the data suggest that Archaea have proteins related to Hop and to the NAC alpha subunit whose functions remain to be elucidated. Other anti-stress means involve osmolytes, ion traffic, and formation of multicellular structures. All cellular anti-stress mechanisms depend on genes whose products are directly involved in counteracting the effects of stressors, or are regulators. The latter proteins monitor and modulate gene activity. Biomethanation depends on the concerted action of at least three groups of microbes, the methanogens being one of them. Their anti-stress mechanisms are briefly discussed in this Chapter from the standpoint of their role in biomethanation with emphasis on their potential for optimizing bioreactor performance. Bioreactors usually contain stressors that come with the influent, or are produced during the digestion process. If the stressors reach levels above those that can be dealt with by the anti-stress mechanisms of the microbes in the bioreactor, the microbes will die or at least cease to function. The bioreactor will malfunction and crash. Manipulation of genes involved in the anti-stress response, particularly those pertinent to the synthesis and regulation of the Hsp70(DnaK) and Hsp60 molecular machines, is a promising avenue for improving the capacity of microbes to withstand stress, and thus to continue biomethanation even when the bioreactor is loaded with harsh waste. The engineering of methanogenic consortia with stress-resistant microbes, made on demand for efficient bioprocessing of stressor-containing effluents and wastes, is a tangible possibility for the near future. This promising biotechnological development will soon become a reality due to the advances in the study of the stress response and anti-stress mechanisms at the molecular and genetic levels.

Archaea↗
PubMed2002

Surface plasmon resonance for measuring TBP-promoter interaction.

A procedure was developed for real-time measurement of the interaction between an archaeal TATA-binding protein (TBP) with stress-gene promoters from the archaeon Methanosarcina mazeii using surface plasmon resonance (SPR), the BIACORE 3000 equipment, and the SA (streptavidin) Sensor Chip. Measurements were based on the SPR optical phenomenon, which resulted in light extinction when TBP bound a promoter. This process, detected as a change in a particular angle, was recorded in a sensorgram. The BIA-evaluation program allowed the calculation of the equilibrium constant (K(A)) of the interaction of M. mazeii TBP with the promoters of the stress genes grpE, hsp70(dnaK), and hsp40(dnaJ) (0.47, 0.26, and 1.21x10(7)M(-1), respectively) and, for comparison, with the promoter of a non-heat-shock gene, orf16 (0.08x10(7)M(-1)). The association rate (k(a)) of the non-heat-shock gene orf16 was 0.4x10(4)M(-1)s(-1) and those for the stress genes, grpE, hsp70(dnaK), and hsp40(dnaJ) were higher: 2.8, 1.5, and 3.5x10(4)M(-1)s(-1), respectively. The new procedure will allow a comparative analysis of different TPBs and promoters (wild type and mutants) under physiologic and stress conditions, and a correlation of TBP binding parameters with constitutive and stress-induced gene expression.

Archaeal Proteins↗
PubMed2002

Effect of heat stress on promoter binding by transcription factors in the cytosol of the archaeon Methanosarcina mazeii.

Regulation of archaeal stress genes is not yet fully understood. This work is part of a research effort aimed at elucidating the molecular mechanisms of transcription initiation and regulation of the stress genes in the hsp70(dnaK) locus of the mesophilic, methanogenic archaeon Methanosarcina mazeii. The locus has the stress genes 5'-grpE-hsp70(dnaK)-hsp40(dnaJ)-3' encoding the chaperone machine components GrpE, Hsp70(DnaK), and Hsp40(DnaJ), respectively, flanked by non-heat shock inducible genes, orf16 and orf11-trkA. Thus, the M. mazeii hsp70(dnaK) locus offers the opportunity for studying heat shock and non-heat shock inducible genes side by side. The objectives of the work reported here were to develop procedures for studying basal transcription factors in the cytosol of M. mazeii and their interaction with these genes' promoters in stressed cells for comparison with unstressed counterparts. The preparation of non-radioactive DNA probes for electrophoretic mobility shift assay (EMSA), and the combination of EMSA with Western blotting for DNA-binding protein identification were standardized for this investigation. DNA probes bearing the genes' promoter regions were used for detecting and identifying DNA-binding proteins in the cytosol of unstressed and heat-shocked cells. Cytosolic TATA-binding protein (TBP) was found to bind the stress-gene promoters in both unstressed and heat-shocked cells but more strongly in the latter. Likewise, in stressed cells TBP-transcription factor B (TFB)(TFIIB) association was increased by comparison with unstressed controls. The level of cytosolic TBP assessed by its DNA-binding activity using EMSA remained unchanged during the various phases of culture growth in the absence of heat stress. The results indicate that heat stress of cells in culture modulates the level and/or the stress-gene promoter-binding activity of the M. mazeii TBP, and enhances TBP-TFB association in the cytosol and DNA binding.

Base Sequence↗
PubMed2002

Sick chaperones and ageing: a perspective.

Intra- and extra-cellular protein deposits characterize many pathologic disorders (proteinopathies). These deposits contain different proteins, some of which are distinctive of a given disorder or type of disease, whereas others are not. The disorder-specific proteins are as a rule defective and have a tendency to misfold, aggregate, and precipitate-thus deposits are formed. The abnormal proteins typical of the better-characterized proteinopathies are not molecular chaperones, and the chaperoning systems in patients with these diseases are apparently normal. In this article, disorders in which abnormal chaperones seem to be the main pathogenetic factor are presented as a subset of the proteinopathies and termed chaperonepathies. A defective chaperone molecule (sick chaperone) will impact on the chaperoning system and pathway in which it normally participates. Since chaperones are ubiquitous their malfunctioning will have universal deleterious effects. In fact, the few known disorders in which a sick chaperone seems to be the main etiology, are clinically heterogeneous with many tissues affected. Senescence is characterized by a progressive decline of vigor and function throughout the body reflecting the failure of many molecules due to wear and tear, accumulation of mutations, and perhaps other factors. It is likely that this age-associated decline is due in part to a progressive failure of the chaperoning systems. Senescence would proceed even faster when a defective chaperone appears early in life due to a genetic defect. Furthermore, sick chaperones will make the individual vulnerable to stressors since the chaperoning systems are key anti-stress mechanisms. In this context, a combination of sick chaperones with stress appears as a driving force in the process of ageing.

Aging↗
PubMed2002

Transcription in the archaea: basal factors, regulation, and stress gene expression.

A brief survey is presented of salient findings on transcription in the Archaea, focussing on stress genes of the hsp70(dnaK) locus, which code for the molecular chaperones Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Archaeal basal factors and some recently characterized regulators pertinent to non-stress genes are presented first to show their similarities and differences with equivalents in organisms of the other two phylogenetic domains, Bacteria and Eucarya, and to reveal clues on how these or similar factors might transcribe and regulate the archaeal stress genes. The second part of the article deals with the hsp70(dnaK)-locus genes, particularly those from Methanosarcina mazeii, because they are virtually the only ones within the methanogenic Archaea whose patterns of constitutive and stress-induced expressions have been studied. Therefore, these genes, provide a standardized model system to elucidate transcription initiation and regulation at the molecular level in this phylogenetic group. Promoters, and other cis-acting sites that are, or might be, involved in stress-gene expression are described. Conformational changes of basal transcription factors after interaction with stress-gene promoters are discussed that suggest ways for generating a large diversity of initiation complexes using a few factors and DNA sites in different combinations. Likewise, the effects of stress on DNA topology and on TBP-TFB-promoter complex formation and tightness are described, which might also contribute to the generation of transcription-initiation complex diversity. This diversity would be key to differential gene expression, namely, which genes are transcribed, when (basal, steady expression vs. sporadic stress-induced expression), and to what level. Future research should investigate this diversity, and the mechanism of complex formation and action at the atomic, molecular, and supramolecular levels to elucidate the dynamics of transcription initiation in real time.

Archaea↗
PubMed2002

Transcription in the archaea: basal factors, regulation, and stress-gene expression.

A brief survey is presented of salient findings on transcription in the Archaea, focussing on stress genes of the hsp70(dnaK locus, which code for the molecular chaperones Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Archaeal basal factors and some recently characterized regulators pertinent to non-stress genes are presented first to show their similarities and differences with equivalents in organisms of the other two phylogenetic domains, Bacteria and Eucarya, and to reveal clues on how these or similar factors might transcribe and regulate the archaeal stress genes. The second part of the article deals with the hsp70(dnaK)-locus genes, particularly those from Methanosarcina mazeii, because they are virtually the only ones within the methanogenic Archaea whose patterns of constitutive and stress-induced expressions have been studied. Therefore, these genes provide a standardized model system to elucidate transcription initiation and regulation at the molecular level in this phylogenetic group. Promoters, and other cis-acting sites that are, or might be, involved in stress-gene expression are described. Conformational changes of basal transcription factors after interaction with stress-gene promoters are discussed that suggest ways for generating a large diversity of initiation complexes using a few factors and DNA sites in different combinations. Likewise, the effects of stress on DNA topology and on TBP-TFB-promoter complex formation and tightness are described, which might also contribute to the generation of transcription-initiation complex diversity. This diversity would be key to differential gene expression, namely, which genes are transcribed, when (basal, steady expression vs. sporadic stress-induced expression), and to what level. Future research should investigate this diversity, and the mechanism of complex formation and action at the atomic, molecular, and supramolecular levels, to elucidate the dynamics of transcription initiation in real time.

Archaea↗
PubMed2002

The genome of M. acetivorans reveals extensive metabolic and physiological diversity.

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic growth. Although motility has not been observed in any Methanosarcineae, a flagellin gene cluster and two complete chemotaxis gene clusters were identified. The availability of genetic methods, coupled with its physiological and metabolic diversity, makes M. acetivorans a powerful model organism for the study of archaeal biology. [Sequence, data, annotations and analyses are available at http://www-genome.wi.mit.edu/.]

Archaeal Proteins↗