Everly Conway de Macario

hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions

BackgroundHsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family.ResultsWe identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD) was not. Nine proteins were typical Hsp70s (65–80 kDa) with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages.ConclusionThe human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-terminal domains likely defining their distinctive functions. These functions may also be further defined by the observed differences in the N-terminal domain.

Hsp60 is actively secreted by human tumor cells

Background Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

Monoclonal antibodies against bacteria.

Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

Isolation and characterization of a thermophilic sulfate reducing archaebacterium archaeoglobus fulgidus strain z

Summary An extremely thermophilic, strictly anaerobic sulfate reducing bacterium, strain Z, was isolated from sediments of a hydrothermal vent located near Mount Vulcanello, Vulcano, Italy. On the basis of 16S rRNA cataloguing, strain Z was shown to belong to the archaebacterial kingdom, representing another strain of Archaeoglobus fulgidus . The weakly motile, irregular coccoid cells showed the fluorescence regarded as typical for methanogens, when viewed under the fluorescence microscope, although no relationship with methanogens was seen from comparative analysis of antigenic fingerprints with antibody S-probes. Furthermore, methane was not produced in significant amounts. Dissimilatory sulfate reduction was found with lactate, pyruvate and 2,3-butandiol as substrates. With thiosulfate as electron acceptor H 2 /CO 2 , formate, lactate, pyruvate or fumarate were utilized as electron donors. Sulfur bloom was not used as electron acceptor. Growth was inhibited by molybdate, a typical inhibitor of sulfate reducers. Lactate was decarboxylated to acetate and part of the acetate was oxidized to CO 2 with the reducing equivalents serving for sulfate reduction. The cell envelope consisted of a hexagonally arranged S-layer. The apparent molecular weight of its subunits, staining PAS-positive, was 132000. No significant amounts of polyamines were detected. The DNA polymerase was sensitive towards aphidicolin, a specific inhibitor of DNA polymerases type a of eucaryotes and of the DNA polymerase of Methanococcus vannielii . The DNA base composition was 45 mol% G+C. Archaeoglobus fulgidus strain Z has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 4139.

Methanogenic archaea in health and disease: a novel paradigm of microbial pathogenesis.

Microbes that produce methane gas, methanogens, were identified as Archaea in the 1970s but their possible role in disease is only emerging now, after they were found in the large intestine, mouth, and vagina. Significant associations were observed, for instance, between levels of methanogens in periodontal pockets and severity of periodontitis, and between quantities of methanogens in the large intestine and diseases such as colon cancer and diverticulosis. Recently, a role for intestinal methanogens in obesity was proposed. The lesson learned is that for methanogens we have to look at their pathogenicity from a different angle in comparison to classic pathogens that invade tissues and release toxins. This type of pathogenicity has not yet been described for methanogens. Instead, methanogens seem to participate in pathogenicity indirectly, favoring the growth of other microbes, which are directly involved in pathogenesis. This indirect role should not be minimized. On the contrary, it has become clear that a fundamental change of approach to the understanding and control of microbial diseases must be implemented. A comprehensive strategy is needed to elucidate the syntrophic associations that are essential for a healthy relation among microbes (including methanogens) and between them and the host organism, and to unveil those associations that lead to disease.

Chlamydia trachomatis infection and anti-Hsp60 immunity: the two sides of the coin.

Chlamydia trachomatis (CT) infection is one of the most common causes of reproductive tract diseases and infertility. CT-Hsp60 is synthesized during infection and is released in the bloodstream. As a consequence, immune cells will produce anti-CT-Hsp60 antibodies. Hsp60, a ubiquitous and evolutionarily conserved chaperonin, is normally sequestered inside the cell, particularly into mitochondria. However, upon cell stress, as well as during carcinogenesis, the chaperonin becomes exposed on the cell surface (sf-Hsp60) and/or is secreted from cells into the extracellular space and circulation. Reports in the literature on circulating Hsp and anti-Hsp antibodies are in many cases short on details about Hsp60 concentrations, and about the specificity spectra of the antibodies, their titers, and their true, direct, pathogenetic effects. Thus, more studies are still needed to obtain a definitive picture on these matters. Nevertheless, the information already available indicates that the concurrence of persistent CT infection and appearance of sf-Hsp60 can promote an autoimmune aggression towards stressed cells and the development of diseases such as autoimmune arthritis, multiple sclerosis, atherosclerosis, vasculitis, diabetes, and thyroiditis, among others. At the same time, immunocomplexes composed of anti-CT-Hsp60 antibodies and circulating Hsp60 (both CT and human) may form deposits in several anatomical locations, e.g., at the glomerular basal membrane. The opposite side of the coin is that pre-tumor and tumor cells with sf-Hsp60 can be destroyed with participation of the anti-Hsp60 antibody, thus stopping cancer progression before it is even noticed by the patient or physician.

Antigenic Fingerprinting of Methanogenic Bacteria with Polyclonal Antibody Probes

A system is described for constructing a reference chart of antigenic fingerprints of methanogens as a possible guide in identification of new isolates. The chart is easy to use and can accommodate into each fingerprint additional data that will be provided by new antisera, and more fingerprints as other reference bacteria will be studied, without changes in its format or accumulated contents. This ensures a perennial force to the chart as a reference instrument, and allows increase of its resolution power for bacterial identification. Based on this chart, a procedure is described for rapid identification of newly isolated methanogens and comparison of antigenic mosaics expressed on their surfaces. The procedure involves determination of the antigenic fingerprint and tabulation of the data for comparison with those in the chart. The main feature of the fingerprinting method is the use of a panel of antibody probes calibrated to reveal dominant antigens. As the probes reveal the most conspicuous markers in the antigenic mosaic they also delineate the most pronounced antigenic relationships among methanogens. Although the methods reported were developed for study of methanogens with polyclonal antibody probes, they can be applied to any other group of organisms with antigenic mosaics and to studies with panels of monoclonal antibodies.

A dnaK homolog in the archaebacterium Methanosarcina mazei S6.

A fragment of genomic DNA cloned from the methanogenic archaebacterium, Methanosarcina mazei strain S6, was found to contain an 1857-bp open reading frame (ORF). A sequence matching the consensus ribosome-binding sequence determined for other methanogens was found upstream from the ORF. The amino acid (aa) sequence encoded by the ORF was compared with reference sequences and was found to be related to six DnaK sequences determined for five species of eubacteria (none exist for archaebacteria). The M. mazei S6 aa sequence was over 61% identical and over 77% similar (identities plus conservative substitutions) to the closest four reference sequences, which were all DnaKs. The gene described here is therefore proposed to be the first member of the dnaK family sequenced from the archaebacterial kingdom (Archaea). This finding confirms that DnaK proteins are highly conserved, occurring not only in eubacteria (Bacteria) and eukaryotes (Eucaria), but also in archaebacteria (Archaea).

The odyssey of Hsp60 from tumor cells to other destinations includes plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities.

Background In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here. Principal Findings We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle. Conclusions/Significance We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

Human Hsp10 and Early Pregnancy Factor (EPF) and their relationship and involvement in cancer and immunity: Current knowledge and perspectives

This article is about Hsp10 and its intracellular and extracellular forms focusing on the relationship of the latter with Early Pregnancy Factor and on their roles in cancer and immunity. Cellular physiology and survival are finely regulated and depend on the correct functioning of the entire set of proteins. Misfolded or unfolded proteins can cause deleterious effects and even cell death. The chaperonins Hsp10 and Hsp60 act together inside the mitochondria to assist protein folding. Recent studies demonstrated that these proteins have other roles inside and outside the cell, either together or independently of each other. For example, Hsp10 was found increased in the cytosol of different tumors (although in other tumors it was found decreased). Moreover, Hsp10 localizes extracellularly during pregnancy and is often indicated as Early Pregnancy Factor (EPF), which is released during the first stages of gestation and is involved in the establishment of pregnancy. Various reports show that extracellular Hsp10 and EPF modulate certain aspects of the immune response with anti-inflammatory effects in patients with autoimmune conditions improving clinically after treatment with recombinant Hsp10. Moreover, Hsp10 and EPF are involved in embryonic development, acting as a growth factor, and in cell proliferation/differentiation mechanisms. Therefore, it becomes evident that Hsp10 is not only a co-chaperonin, but an active player in its own right in various cellular functions. In this article, we present an overview of various aspects of Hsp10 and EPF as they participate in physiological and pathological processes such as the antitumor response and autoimmune diseases.