Major Classes Of Proteins Research Articles

The small heat shock proteins of the nematode Caenorhabditis elegans: structure, regulation and biology.

The first investigations of the heat shock response in C. elegans were carried out by analyzing the patterns of proteins synthesized by 35S-labeled nematodes cultured at various temperatures (Snutch and Baillie 1983). These experiments established a temperature profile for the heat shock response, identified the major molecular weight classes of heat shock proteins (Hsps) and noted the homology between Drosophila and C. elegans Hsp70 genes, based on crosshybridization experiments. In these studies, a prominent, labeled protein with an apparent molecular mass of 16 kDa was noted in heat shocked animals. Partial cDNAs encoding Hspl6 species were subsequently cloned, and these were the first small Hsp (sHsp) sequences determined after those of Drosophila, to which they were found to be related (Russnak et al. 1983). The Hsp 16 sequences of C. elegans thus provided the first indication that small Hsps of varying molecular masses from distantly related organisms were similar to each other and to the mammalian α-crystallins. The completion of the C. elegans genome sequence (The C. elegans Sequencing Consortium 1998) provides an opportunity to examine the entire complement of sHsps from this metazoan. This review will provide an overview of the structures, expression, developmental and tissue specificities and biochemical properties of the C. elegans sHsps which have been studied to date.

The impact of multiple drug resistance (MDR) proteins on chemotherapy and drug discovery.

Transportation of molecules across the cell membrane in living organisms is a critical aspect of life. Transportation includes importation of nutrients from the environment and exportation of toxic compounds. When export includes therapeutic compounds, then the practice of clinical medicine may become compromised. Often efflux of therapeutic compounds is mediated by a large superfamily of proteins referred to as multidrug resistance (MDR) proteins. The initial sections of this chapter are focused on MDR proteins and their negative impact on clinical medicine in cancer chemotherapy as well as infectious diseases mediated by bacteria, fungi and parasites. A brief description of major classes of MDR proteins found in microbes is followed by a more exhaustive treatment of ABC transporters in lower eukaryotes and parasites as well as cancerous mammalian cells. Later sections deal with potential and real positive aspects and applications brought about by a growing knowledge of MDR proteins. Examples described include improved antibiotic production, leveraging MDR proteins in drug discovery, new therapeutic options, dual therapy in treatment of cancer and infectious diseases, and finally MDR proteins as targets for new classes of therapeutic compounds.

Nuclear transport of photosensitizers during photosensitization and oxidative stress

The nuclear transport pathways of the photosensitizers meso-tetra(4-sulfonatophenyl)porphyrin (TPPS 4) and meso-tetra(4-N-methylpyridyl)porphyrin (TMPyP) during photosensitization and oxidative stress were characterized in CT-26 murine colon carcinoma cells using fluorescence microscopy and multi-pixel spectral imaging. Prior to irradiation, TPPS 4 and TMPyP localized mainly in the lysosomes, while irradiation or H 2O 2 treatment induced a relocalization into the nucleus and nucleoli. Flow cytometry analysis of isolated nuclei from the treated cells showed an increase in nuclear fluorescence accompanying the relocalization. Isolation and separation of the nuclear proteins according to molecular weight was performed using a sephadex G-100 column. The protein fractions exhibiting high fluorescence were separated by high performance liquid chromatography. Five major classes of proteins with a retention time of 1, 7, 11, 12 and 15 min were obtained. Each photosensitizer was associated with a distinct class of proteins. While TPPS 4 fluorescence was detected in the protein fraction with a retention time of 11 min, TMPyP fluorescence was associated with a protein fraction having a retention time of 7 min. We conclude that although oxidative stress triggers entry into the nucleus of both TPPS 4 and TMPyP, each sensitizer uses a distinct transport mechanism based on its chemical properties.

Towards the molecular mechanism of prokaryotic and eukaryotic multidrug transporters

Due to their ability to extrude structurally dissimilar cytotoxic drugs out of the cell, multidrug transporters are able to reduce the cytoplasmic drug concentration, and, hence, are able to confer drug resistance on human cancer cells and pathogenic microorganisms. This review will focus on the molecular properties of two bacterial multidrug transporters, the ATP-binding cassette transporter LmrA and the proton motive force-dependent major facilitator superfamily transporter LmrP, which each represent a major class of multidrug transport proteins encountered in pro- and eukaryotic cells. In spite of the structural differences between LmrA and LmrP, the molecular bases of their drug transport activity may turn out to be more similar than might currently appear.

Genetic variations and polymorphisms of G protein-coupled receptors: functional and therapeutic implications.

G protein-coupled receptors (GPCRs) represent a major class of proteins in the genome of many species, including humans. In addition to the mapping of a number of human disorders to regions of the genome containing GPCRs, a growing body of literature has documented frequently occurring variations (i.e. polymorphisms) in GPCR loci. In this article, we use a domain-based approach to systematically examine examples of genetic variation in the coding and noncoding regions of GPCR loci. Data to date indicate that residues in GPCRs are involved in ligand binding and coupling to G proteins and that regulation can be altered by polymorphisms. Studies of GPCR polymorphisms have also uncovered the functional importance of residues not previously implicated from other approaches that are involved in the function of GPCRs. We predict that studies of GPCR polymorphisms will have a significant impact on medicine and pharmacology, in particular, by providing new means to subclassify patients in terms of both diagnosis and treatment.

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.

Seed storage proteins in cultivars and subspecies of alfalfa (Medicago sativa L.)

AbstractSeed storage proteins were analysed in 27 varieties of alfalfa (Medicago sativaL.); these included five subspecies (glomerata, caerulea, falcata, hemicycla, praefalcata), seven of the nine sources of Medicago germplasm introduced into North America and a sample of additional cultivars. The protein patterns were remarkably consistent for all of these taxa. One-dimensional and two-dimensional gel electrophoresis revealed only minor differences in polypeptide composition within each of the three major classes of storage protein (7S globulin, 11S globulin, 2S albumin). The slight variations that were found provided no information on either parentage or evolutionary relationships amongst these particular taxa. Nonetheless, persistent and reproducible heterogeneity of some minor polypeptides of 11S globulin (medicagin) may be useful under other circumstances for cultivar identification in alfalfa. Both subfamilies (I and II) of the 11S globulin were strongly expressed in all of the cultivars and subspecies examined. It was concluded that this structural divergence within the 11S storage protein family predated the evolution of the M. sativa L. species complex. Most of the variability in storage proteins was quantitative. However, even this variability was reduced when data were standardized with respect to seed dry weights. The consistent similarities in qualitative and quantitative expression of seed storage proteins amongst all of these taxa suggest a high degree of uniformity in both seed physiology and genetics within the alfalfa species complex.

Regulation of Cyclin-Dependent Kinase 2 Activity by Ceramide

Cyclin-dependent kinases have been implicated in the inactivation of retinoblastoma (Rb) protein and cell cycle progression. Recent studies have demonstrated that the lipid molecule ceramide is able to induce Rb hypophosphorylation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C6-Ceramide induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblasts. Employing immunoprecipitation kinase assays, we found that ceramide specifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C6-dihydroceramide was not effective. Ceramide did not directly inhibit CDK2 in vitro but caused activation of p21, a major class of CDK-inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both protein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 in vitro. Further, calyculin A and okadaic acid, both potent protein phosphatase inhibitors, together almost completely reversed the effects of ceramide on CDK2 inhibition. Taken together, these results demonstrate a dual mechanism by which ceramide inhibits the cell cycle. Ceramide causes an increase in p21 association with CDK2 and through activation of protein phosphatases selectively regulates CDK2. These events may lead to activation of Rb protein and subsequent cell cycle arrest.

Evidence for two distinct classes of high affinity growth hormone binding proteins in pregnant rat serum.

These studies have established the presence of two major classes of high affinity growth hormone binding proteins in pregnant rat serum, designated GHBPa and GHBPb, with apparent native Mr of 257 K and 98 K respectively. GHBPa, which has not been identified previously, exhibits a binding affinity (2-5 nM(-1)) that is up to 20-fold higher than GHBPb (0.2-0.8 nM(-1)) and is the least abundant form, being approximately 15-20% of total serum GH-binding capacity. Western immunoblot analysis revealed that each GHBP is composed of several immunoreactive proteins which were reactive with carboxy-terminal (RB1615) and/or N-terminal (MAb263) domain antibodies, suggesting the presence of GHBPs with and without the hydrophilic tail. Of importance is that GHBPa exhibited significantly higher Mr (78-182 K, +DTT) than that predicted by GHBP cloning, suggesting that they may be covalently bound to other non-GH-binding proteins or may be distinct entities. GHBPb, on the other hand, was composed of smaller Mr (43/48 K, +DTT) "hydrophilic" tail-containing proteins, some of which were disulphide linked to a larger complex of approximately 110 K. These novel findings challenge the current view of the mechanism for generation of the rat serum GHBP and raise the intriguing possibility that the two classes of GHBP may play distinct and important roles in GH physiology.

Stage specific kinetoplast DNA-binding proteins in Trypanosoma cruzi

Knowledge regarding kinetoplast DNA organization in all members of the Trypanosomatid family is incomplete. Recently, the presence of kinetoplast-associated proteins in condensing kDNA networks in Crithidia fasciculata has been described and a role for these proteins in the maintenance of these complex structures was suggested. To investigate the presence of protein components in Trypanosoma cruzi kinetoplast, we previously described seven epimastigote kinetoplast-associated proteins. We report here the existence of kinetoplast binding proteins in amastigote and trypomastigote stages of T. cruzi, which could bind both mini and maxicircles components with a stage specific elements for every infective form of the parasite. We propose three major classes of kinetoplast-associated proteins related to the basic processes of this intricate disc structure and suggest a possible function of these binding proteins in the T. cruzi mitochondrial DNA organization.

Paramecium GPI Proteins: Variability of Expression and Localization

In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 degrees C and 32 degrees C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 degrees C and 32 degrees C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region.

Splice variants of the nuclear dot-associated Sp100 protein contain homologies to HMG-1 and a human nuclear phosphoprotein-box motif.

Sp100 and PML are interferon-inducible proteins associated with a new class of nuclear domains (known as nuclear dots or PML bodies) which play a role in tumorigenesis, virus infections, and autoimmunity. While PML is extensively alternatively spliced, only two splice variants are known for Sp100. Here we describe the identification and characterization of several Sp100 splice variant proteins and support their existence by elucidation of the 3'-end of the Sp100 gene. Some of the splice variants contain a domain of significant sequence similarity with two previously described highly related interferon-inducible nuclear phosphoproteins as well as to suppressin and DEAF-1, which altogether define a novel protein motif, termed HNPP-box. One class of splice variants contains an almost complete and highly conserved copy of the DNA-binding high mobility group 1 protein sequence and thus represent novel HMG-box proteins. When expressed transiently, both major classes of Sp100 splice variant proteins localize in part to nuclear dots/PML bodies and in addition to different nuclear domains. Furthermore, PML was occasionally redistributed. These data indicate that alternatively spliced Sp100 proteins are expressed, differ in part in localization from Sp100, and might bind to chromatin via the HMG domain.

15] Reconstitution of native and cloned channels into planar bilayers

A planar bilayer is an artificial membrane formed across a small hole, ∼50 μm or larger in diameter. The hole on which the membrane is formed is usually placed in a thin plastic partition separating two aqueous compartments, but artificial bilayers may also be formed on a glass micropipette tip. Insertion or incorporation of a channel-forming molecule into such a membrane provides a simple experimental system for electrical recording of channel-mediated currents. Planar bilayer recording of ion channels is practiced for a number of reasons. It is a technique that yields incredibly rich mechanistic information on a relatively low budget while offering kaleidoscopic displays of single-channel fluctuations that some workers find delightful, even soothing.. This chapter highlights technical aspects of selected examples where the bilayer approach has been widely applied to functional analysis of major classes of ion channel proteins. Identification of techniques and preparations that have been found to be most generally applicable may facilitate extension of the bilayer approach to other classes of channel proteins that have not yet been thoroughly domesticated.

Activation of three classes of nonreceptor tyrosine kinases following Fc gamma receptor crosslinking in human monocytes.

Fc gamma receptors on monocytes/macrophages play an important role in both host defense and autoimmune disorders. Fc gamma receptor signaling can lead to such downstream events as phagocytosis and the release of intracellular cytokines and reactive oxygen species. Freshly isolated human monocytes express two major classes of Fc gamma receptor proteins, Fc gamma RI (CD64) and Fc gamma RII (CD32). Crosslinking of Fc gamma RI and Fc gamma RII gives rise to rapid and transient phosphorylation of multiple monocyte intracellular proteins including proteins of 40, 68-72, 75-85, 95, and 115-165 kDa. A 72-kDa protein was earlier identified as the tyrosine kinase Syk. Here we identify one of the proteins in the 115- to 165-kDa cluster as FAK, a protein tyrosine kinase localized to focal adhesions. A 68-kDa phosphoprotein was identified as paxillin, a cytoskeleton associated substrate for tyrosine kinases, and a 95-kDa protein was found to be the proto-oncogene product Vav. The Src family protein tyrosine kinase Fgr (p58) also displayed enhanced tyrosine phosphorylation after Fc gamma RI and Fc gamma RII crosslinking. Although Fc gamma RIIA utilizes tyrosines within its own cytoplasmic domain for signaling while Fc gamma RI utilizes the cytoplasmic tyrosines of its associated gamma subunit, our results indicate sharing of several proteins for signaling in monocytes by these Fc receptors. These molecules include three distinct classes of tyrosine kinases, Syk, FAK, and Fgr, and the functionally diverse proteins Vav and paxillin.

Light-Regulated Transcription of Genes Encoding Peridinin Chlorophyll a Proteins and the Major Intrinsic Light-Harvesting Complex Proteins in the DinoflagellateAmphidinium carterae Hulburt (Dinophycae)1

In the dinoflagellate Amphidinium carterae, photoadaptation involves changes in the transcription of genes encoding both of the major classes of light-harvesting proteins, the peridinin chlorophyll a proteins (PCPs) and the major a/c-containing intrinsic light-harvesting proteins (LHCs). PCP and LHC transcript levels were increased up to 86- and 6-fold higher, respectively, under low-light conditions relative to cells grown at high illumination. These increases in transcript abundance were accompanied by decreases in the extent of methylation of CpG and CpNpG motifs within or near PCP- and LHC-coding regions. Cytosine methylation levels in A. carterae are therefore nonstatic and may vary with environmental conditions in a manner suggestive of involvement in the regulation of gene expression. However, chemically induced undermethylation was insufficient in activating transcription, because treatment with two methylation inhibitors had no effect on PCP mRNA or protein levels. Regulation of gene activity through changes in DNA methylation has traditionally been assumed to be restricted to higher eukaryotes (deuterostomes and green plants); however, the atypically large genomes of dinoflagellates may have generated the requirement for systems of this type in a relatively "primitive" organism. Dinoflagellates may therefore provide a unique perspective on the evolution of eukaryotic DNA-methylation systems.

Stability of Hor1-specific YAC-clones and physical mapping of Hor1-loci in barley

The hordeins are the major class of storage proteins in barley and are encoded by multigene families. Two YAC-clones specific for the C-hordein-coding Hor1-locus of barley (Hordeum vulgare L.) were selected. The clones were constructed with DNA from the cultivars ‘Franka’ and ‘Hockey’ and have insert sizes of 330 kb and 350 kb, respectively. Performing partial digestions and hybridizations with vector-specific probes, a restriction analysis was conducted using restriction enzymes with a 8-bp recognition sequence. Both clones cover the complete region of the Hor1-locus, but exhibit a different pattern of restriction sites reflecting the polymorphic nature of the locus on the scale of long-range restriction mapping. The maximal extent of the regions homologous to the Hor1-specific probe, pBSC5, was 105 kb in the ‘Hockey’-derived YAC and 190 kb in the yeast artificial chromosome constructed with ‘Franka’-DNA. Furthermore the high degree of instability observed with the Hor1-specific YAC-clones is discussed in conjunction with the structure of the Hor1-locus.

The activation domain of the maize transcription factor Opaque-2 resides in a single acidic region.

The maize (Zea mays L.) endosperm specific transcription factor, encoded by the Opaque-2(O2) locus, functions in vivo to activate transcription from its target promoters.O2 regulates the expression of a major storage protein class, the 22 kDa zeins, and of a type I ribosome inactivating protein, b-32, during maturation phase endosperm development. The coding sequence of O2, which indicates it to be a member of the basic region-leucine zipper (bZIP) class of DNA-binding proteins, contains a number of regions rich in either proline or acidic residues which are candidates for activation domains. In functional assays using tobacco mesophyll protoplasts, the level of transactivation conferred by a series of O2-deletion constructs was tested using as a reporter a fusion of the b-32 target promoter to beta-glucuronidase (GUS). The results indicate that O2 has a single acidic activation domain, located near the N-terminus of the protein (amino acids 41-91). The ability of a shorter part of this domain (amino acids 39-82) to confer transactivation was also demonstrated in domain swapping experiments, using fusions of the O2 polypeptide sequence to the DNA-binding domain of the parsley (Petroselinum crispum) transcription factor CPRF1.

Timing of the expression of enamel gene products during mouse tooth development.

In order to understand the mechanisms involved in tooth development it is important to define the timing for tissue-specific gene expression. A consequence of ameloblast cell differentiation is the sequential expression of tissue-specific genes whose products form the enamel extracellular matrix. The ameloblast phenotype has been characterized as consisting of two major classes of proteins: amelogenins and non-amelogenin proteins such as anionic enamel proteins (enamelins, tuft proteins, tuftelin, sulfated proteins) and enamel proteases. The postulated functions for the anionic enamel proteins are as nucleators for hydroxyapatite crystal formation while amelogenins control the crystal size, growth and orientation. While the amelogenins have been well characterized, detailed knowledge for anionic enamel proteins has been sparse. In the present study, we designed experiments to characterize one of the anionic enamel proteins from mouse molars, tuftelin, and to determine the timing of expression of this protein during molar tooth development. Our results showed the initial detection of tuftelin transcripts within proliferating inner enamel epithelial cells at very early stages of tooth development (13 days of embryonic development equivalent to the bud stage of tooth development). These data provide direct evidence that invalidates previous dogmas that enamel proteins were synthesized by polarized, non-dividing, fully differentiated ameloblast cells. In addition, tuftelin was found to be synthesized also by dental papilla mesenchyme cells suggesting that this protein is not enamel-specific. These data taken together open the possibility that the tuftelin present in the dentino-enamel junction could be secreted by both, preodontoblast cells and preameloblast cells. It might also suggest a possible different role for tuftelin than nucleator of hydroxyapatite crystals.

The Effects of Ozone on Antioxidant Responses in Plants

The response of plants to ozone exposure includes a number of physiological and biochemical changes that are the direct result of selective increases or decreases in gene expression and the resulting changes in the accumulation of the corresponding protein products. Major classes of ozone-induced proteins include antioxidant enzymes and a number of stress-related proteins associated with other biotic and abiotic stresses. In particular, there is a significant overlap in the pattern of gene induction observed in ozone-treated plants and plants exhibiting pathogen defense responses. Current knowledge concerning the specific molecular events associated with the alterations of gene expression caused by ozone and the precise roles of ozone-induced proteins in conferring tolerance to ozone is rather limited. This review summarizes some of the recent results that have been obtained concerning the molecular basis of ozone-induced responses in plants, with an emphasis on studies of the model plant system, Arabidopsis thaliana. These studies demonstrate that ozone-induced responses are caused in part by the activation of a salicylic acid dependent signaling pathway that is also required for the expression of resistance to microbial pathogens.

Intermediate filaments in health and disease

lntermediate filaments constitute one of the major classes of cytoskeletal proteins of mammalian cells. The 40 or more known intermediate filament proteins have been classified into five types which show very specific rules of expression in specialized cell types, and include: type I and type II keratins of epithelial cells; type III vimentin (mesenchymal cells), desmin (muscle), glial fibrillary acidic protein (astroglia), and peripherin (peripheral neurones); type IV neurofilaments of neuronal cell types; and type V nuclear lamins. lntermediate filaments differ from ubiquitous microfilaments (actin) and microtubules (tubulin) in several important ways, including their structures, intracellular dynamics, and functions. Notably, intermediate filament chains possess a characteristic central α-helical domain that forms coiled-coil rods which in turn assemble into 10-15 nm diameter filaments. The end domains which flank the rod domain are highly variable in sequence and confer specific functions to the filaments in different cell types. Recently, a number of diseases have been identified in human that involve mutations in genes encoding intermediate filament chains. These include seven different keratin diseases, usually caused by mutations in rod domain sequences which often result in serious skin (epidermal) abnormalities; and many cases of sporadic amyotrophic lateral sclerosis caused by mutations in the end domain sequences of neurofilament chains.