The reticulons: a family of proteins with diverse functions

The reticulon family is a large and diverse group of membrane-associated proteins found throughout the eukaryotic kingdom. All of its members contain a carboxy-terminal reticulon homology domain that consists of two hydrophobic regions flanking a hydrophilic loop of 60-70 amino acids, but reticulon amino-terminal domains display little or no similarity to each other. Reticulons principally localize to the endoplasmic reticulum, and there is evidence that they influence endoplasmic reticulum-Golgi trafficking, vesicle formation and membrane morphogenesis. However, mammalian reticulons have also been found on the cell surface and mammalian reticulon 4 expressed on the surface of oligodendrocytes is an inhibitor of axon growth both in culture and in vivo. There is also growing evidence that reticulons may be important in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. The diversity of structure, topology, localization and expression patterns of reticulons is reflected in their multiple, diverse functions in the cell.     

ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions.

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries.     

CHD proteins: a diverse family with strong ties.

Chromodomain/helicase/DNA-binding domain (CHD) proteins have been identified in a variety of organisms. Despite common features, such as their chromodomain and helicase domain, they have been described as having multiple roles and interacting partners. However, a common theme for the main role of CHD proteins appears to be linked to their ATP-dependent chromatin-remodeling activity. Their actual activity as either repressor or activator, and their cell or gene specificity, is connected to their interacting partner(s). In this minireview, we attempt to match the members of the CHD family with the presence of structural domains, cofactors, and cellular roles in the regulation of gene expression, recombination, genome organization, and chromatin structure, as well as their potential activity in RNA processing.     

Thrombospondin 2, a matricellular protein with diverse functions.

Thrombospondin (TSP) 2 is a close relative of TSP1 but differs in its temporal and spatial distribution in the mouse. This difference in expression undoubtedly reflects the marked disparity in the DNA sequences of the promoters in the genes encoding the two proteins. The synthesis of TSP2 occurs primarily in connective tissues of the developing and growing mouse. In the adult animal the protein is again produced in response to tissue injury and in association with the growth of tumors. Despite the abnormalities in collagen fibrillogenesis, fragility of skin, and laxity of tendons and ligaments observed in the TSP2-null mouse, TSP2 does not appear to contribute directly to the structural integrity of connective tissue elements. Instead, emerging evidence supports a mode of action of TSP2 'at a distance', i.e. by modulating the activity and bioavailability of proteases and growth factors in the pericellular environment and, very likely, by interaction with cell-surface receptors. Thus, TSP2 qualifies as a matricellular protein, as defined in the introduction to this minireview series. The phenotype of TSP2-null mice has been very helpful in providing clues to the functions of TSP2. In addition to histological and functional abnormalities in connective tissues, these mice display an increased vascularity of the dermis and subdermal tissues, increased endosteal bone growth, a bleeding defect, and a marked adhesive defect of dermal fibroblasts. Our laboratory has established that TSP2 binds matrix metalloproteinase 2 (MMP2) and that the adhesive defect in TSP2-null fibroblasts results from increased MMP2 activity. The investigation of the basis for the other defects in the TSP2-null mouse is likely to yield equally interesting results.     

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.     

Secreted inducers in vertebrate eye development: more functions for old morphogens

Cell signaling molecules secreted from strategically localized positions coordinate cell behavior to enable progressive specification of embryonic tissues. These molecules converge on a few signaling pathways that are reiteratively used in different tissues at different times for generating cell type-specific patterns of gene expression. Although our current knowledge of the system is fragmentary, eye development seems to follow this general strategy. In line with this idea, recent studies have added new information on how Fgf and Wnt signaling participates in the formation of the eye field. In addition, later on in development, Fgf controls the onset of retinal neurogenesis and Shh and GDF11 control its feedback regulation.     

The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions

The human heat shock protein 70 (Hsp70) family contains at least eight homologous chaperone proteins. Endoplasmatic reticulum and mitochondria have their specific Hsp70 proteins, whereas the remaining six family members reside mainly in the cytosol and nucleus. The requirement for multiple highly homologous although different Hsp70 proteins is still far from clear, but their individual and tissue-specific expression suggests that they are assigned distinct biological tasks. This concept is supported by the fact that mice knockout for different Hsp70 genes display remarkably discrete phenotypes. Moreover, emerging data suggest that individual Hsp70 proteins can bring about non-overlapping and chaperone-independent functions essential for growth and survival of cancer cells. This review summarizes our present knowledge of the individual members of human Hsp70 family and elaborate on the functional differences between the cytosolic/nuclear representatives.     

The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes

Phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs) remain largely functionally uncharacterized, despite the fact that they are highly conserved and are found in all eukaryotic cells thus far examined by biochemical or sequence analysis approaches. The available data indicate a role for PITPs in regulating specific interfaces between lipid-signaling and cellular function. In this regard, a role for PITPs in controlling specific membrane trafficking events is emerging as a common functional theme. However, the mechanisms by which PITPs regulate lipid-signaling and membrane-trafficking functions remain unresolved. Specific PITP dysfunctions are now linked to neurodegenerative and intestinal malabsorption diseases in mammals, to stress response and developmental regulation in higher plants, and to previously uncharacterized pathways for regulating membrane trafficking in yeast and higher eukaryotes, making it clear that PITPs are integral parts of a highly conserved signal transduction strategy in eukaryotes. Herein, we review recent progress in deciphering the biological functions of PITPs, and discuss some of the open questions that remain.     

Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66–68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX–Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX–Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca²⁺ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX–Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.     

Ferritin: an iron storage protein with diverse functions

Ferritin is the major protein for iron storage and iron detoxification. Since non-ferrous metals, such as aluminum, beryllium and zinc, are bound both in vivo and in vitro, ferritin is implicated as a general metal ion donor and detoxicant. The role of ferritin in Al and Be toxicity is discussed. During iron release ferritin produces free radicals which are involved in phosphoprotein inactivation, lipid peroxidation and, possibly, the general aging process. Conversely, during iron loading oxidative energy in the form of electrons and protons is given off. The different subunit compositions of ferritin, termed isoferritins, are, at least in part, involved with the multifunctionality of this protein.     

GMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities.

Sequence comparison of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase and Hansenula polymorpha methanol oxidase indicates that these four diverse flavoproteins are homologous, defining a new family of proteins named the GMC oxidoreductases. These enzymes contain a canonical ADP-binding beta alpha beta-fold close to their amino termini as found in other flavoenzymes. This domain is encoded by a single exon of the D. melanogaster glucose dehydrogenase gene.     

Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins

The FAM69 family of cysteine-rich type II transmembrane proteins comprises three members in all vertebrates except fish, and orthologues with a conserved structure are present throughout metazoa. All three murine FAM69 proteins (FAM69A, FAM69B, FAM69C) localise to the endoplasmic reticulum (ER) in cultured cells, probably via N-terminal di-arginine motifs. Mammalian FAM69A is ubiquitously expressed, FAM69B is strongly expressed in the brain and in peripheral endothelial cells, and FAM69C in the brain and eye. Antibodies against mouse FAM69B strongly stain the ER of a subset of neurons in the brain. FAM69 proteins are likely to play a fundamental and highly conserved role in the ER of most metazoan cells, with additional specialised roles in the vertebrate nervous system.     

Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins

Myo-inositol is one of the major organic osmolytes in the brain and the kidney. The accumulation of intracellular organic osmolytes allows cells to regulate intracellular osmolality without altering cytoplasmic ionic strength and to adapt to hyperosmotic conditions. Two types of myo-inositol transporters, sodium/myo-inositol transporter and H⁺/myo-inositol transporter (HMIT), have been identified. Sodium/myo-inositol transporters are induced by osmotic stress and might be involved in the intracellular accumulation of myo-inositol in mammals. The role of HMIT, however, remains unknown. In the present study, we characterized three Caenorhabditis elegansHMIT genes, hmit-1.1, hmit-1.2, and hmit-1.3. hmit-1.1 was expressed in the intestine, and hmit-1.2 was expressed in the glia and the excretory canal, which is an osmotic regulatory organ that is functionally analogous to the kidney. hmit-1.3 was expressed in the intestine and the glia. The expression of hmit-1.1 and hmit-1.2 but not hmit-1.3, was markedly induced under hyperosmotic conditions. Animals with mutant hmit-1.1 and hmit-1.2 were hypersensitive to osmotic stress. The defects of hmit-1.1 and hmit-1.2 mutants were rescued by hmit-1.1 and hmit-1.2 transgenes, respectively, and by modified human HMIT. In human cell lines, HMIT expression was induced in hyperosmotic conditions. These findings indicate that the C. elegans HMIT family has a crucial role in the osmoprotective response.     

All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.