Metallothionein protein evolution: a miniassay

Metallothionein (MT) evolution is one of the most obscure yet fascinating aspects of the study of these atypical metal-binding peptides. The different members of the extremely heterogeneous MT protein superfamily probably evolved through a web of duplication, functional differentiation, and/or convergence events leading to the current scenario, which is particularly hard to interpret in terms of molecular evolution. Difficulties in drawing straight evolutionary relationships are reflected in the lack of definite MT classification criteria. Presently, MTs are categorized either according to a pure taxonomic clustering or depending on their metal binding preferences and specificities. Extremely well documented MT revisions were recently published. But beyond classic approaches, this review of MT protein evolution will bring together new aspects that have seldom been discussed before. Hence, the emergence of life on our planet, since metal ion utilization is accepted to be at the root of the emergence of living organisms, and global trends that underlie structural and functional MT diversification, will be presented. Major efforts are currently being devoted to identifying rules for function-constrained MT evolution that may be applied to different groups of organisms.     

Endophilin-1: a multifunctional protein

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.     

RBM4: a multifunctional RNA-binding protein

RBM4, also known as Lark, was described initially as having a role in circadian rhythm control in Drosophila. In the last 5 years data have emerged from studies of mammalian cells. It is now clear that RBM4 is an RNA-binding protein involved in diverse cellular processes that include alternative splicing of pre-mRNA, translation, and RNA silencing. Its structure, similar to other RNA-binding proteins, contains two RNA recognition motifs and a CCHC-type zinc finger. Here we review current information about the function of RBM4 and its localization within the cell. We then speculate about its possible relationship to disease.     

Cinnamomin: a multifunctional type II ribosome-inactivating protein

Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes by specifically removing the conserved adenine base from the "Sarcin/Ricin domain" of the 28S RNA in ribosome. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of camphor tree. Besides site-specific deadenylation of the A4324 in the Sarcin/Ricin domain of rat ribosome, this protein could also release the adenine base from DNA molecules at multiple sites and from AMP, ADP, dAMP and adenosine. Furthermore, cinnamomin displays cytotoxicity to carcinoma cells and insect larvae by modifying their ribosomal RNA. These functions possessed by cinnamomin shed a new light on the possible application of cinnamomin in the field of immunotoxin design and transgenic reagents. In this review, we introduce the major recent results on cinnamomin obtained in our laboratory, including purification of this protein, characterization of its enzymatic mechanism, structure and function, gene pattern, physiological role and its biological implications in cytotoxicity.     

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.     

RIG-I: a multifunctional protein beyond a pattern recognition receptor

It was widely known that retinoic acid inducible gene I (RIG-I) functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs. However, recent studies showed that RIG-I participates in other various cellular activities by sensing endogenous RNAs under different circumstances. For example, RIG-I facilitates the therapy resistance and expansion of breast cancer cells and promotes T cell-independent B cell activation through interferon signaling activation by recognizing non-coding RNAs and endogenous retroviruses in certain situations. While in hepatocellular carcinoma and acute myeloid leukemia, RIG-I acts as a tumor suppressor through either augmenting STAT1 activation by competitively binding STAT1 against its negative regulator SHP1 or inhibiting AKT-mTOR signaling pathway by directly interacting with Src respectively. These new findings suggest that RIG-I plays more diverse roles in various cellular life activities, such as cell proliferation and differentiation, than previously known. Taken together, the function of RIG-I exceeds far beyond that of a pattern recognition receptor.     

The agnoprotein of polyomaviruses: a multifunctional auxiliary protein

The late region of the three primate polyomaviruses (JCV, BKV, and SV40) encodes a small, highly basic protein known as agnoprotein. While much attention during the last two decades has focused on the transforming proteins encoded by the early region (small and large T-antigens), it has become increasingly evident that agnoprotein has a critical role in the regulation of viral gene expression and replication, and in the modulation of certain important host cell functions including cell cycle progression and DNA repair. The importance of agnoprotein is underscored by its expression during lytic infection of glial cells by JCV that occurs in progressive multifocal leukoencephalopathy (PML), and also in some JCV-associated human neural tumors particularly medulloblastoma. In this review, we will discuss the structure and function of agnoprotein in the viral life cycle during the course of lytic infection and the consequences of agnoprotein expression for the host cell.     

Galectin-3 as a multifunctional protein

Galectin-3 is a 31 kDa member of a growing family of beta-galactoside-binding animal lectins. This protein is expressed in a variety of tissues and cell types and is mainly found in the cytoplasm, although, depending on cell type and proliferative state, a significant amount of this lectin can also be detected in the nucleus, on the cell surface or in the extracellular environment. Galectin-3 is secreted from cells by a novel and incompletely understood mechanism that is independent of the classical secretory pathway through the endoplasmic reticulum/Golgi network. Galectin-3 exhibits pleiotropic biological function, playing a key role in many physiological and pathological processes.     

Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [3H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. We propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment.     

Secretion of prosaposin, a multifunctional protein, by breast cancer cells.

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.     

Metallothionein as a prognostic biomarker in breast cancer

Breast cancer is the most common cancer in women, with a general upward trend in incidence. Basic and clinical breast cancer research has continued at a rapid pace, in the endeavor to understand the biology of the disease so as to improve management of patients. Besides traditional pathological indicators, expression of molecular markers in breast cancer has also been comprehensively investigated. This paper will focus on the prognostic utility of metallothioneins (MTs), a family of low molecular weight metal binding proteins encoded by at least 10 functional MT genes that are associated with cell proliferation in breast cancer. Evidence that MT is a potential prognostic biomarker for breast cancer is supported by many reports in the literature. Expression of the MT protein has been detected by immunohistochemistry in a significant portion of invasive ductal breast cancers. MT expression has also been well studied in association with traditional clinico-pathological parameters of breast cancers. Generally, higher MT expression in breast cancers is predictive of worse patient outcomes. The relationship of MT isoforms to histological grade, estrogen receptor (ER) status, and prognosis will also be discussed.     

Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

The E protein is a multifunctional membrane protein of SARS-CoV

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.