The chaotrope-soluble glycoprotein GP2 is a precursor of the insoluble glycoprotein framework of the Chlamydomonas cell wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.     

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.     

14-3-3 proteins are involved in the regulation of mammalian cell proliferation

Summary. The 14-3-3 proteins are a family of abundant, widely expressed acidic polypeptides. The seven isoforms interact with over 70 different proteins. 14-3-3 isoforms have been demonstrated to be involved in the control of positive as well as negative regulators of mammalian cell proliferation. Here we used the approach of inactivating 14-3-3 protein functions via overexpression of dominant negative mutants to analyse the role of 14-3-3 proteins in mammalian cell proliferation. We found 14-3-3 dominant negative mutants to downregulate the proliferation rates of HeLa cells. Overexpression of these dominant negative mutants triggers upregulation of the protein levels of the cyclin-dependent kinase inhibitor p27, a major negative cell cycle regulator. In addition, they downregulate the protein levels of the important cell cycle promoter cyclin D1. These data provide new insights into mammalian cell proliferation control and allow a better understanding of the functions of 14-3-3 proteins.     

14-3-3s are DNA-replication proteins

14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes. Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family. As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication. Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes. The involvement of cruciform structures in the initiation of DNA replication has been demonstrated. A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes.     

Multifunctional 14-3-3 proteins of plant cell

The 14-3-3 proteins are a family of highly conserved proteins found in all eukaryotes - from the yeasts to mammals. They regulate several cellular processes recognizing unique conservative, mostly phosphorylated motif of partner proteins. Binding of the 14-3-3 proteins regulates their partners through a variety of mechanisms, such as altering their catalytic activity, subcellular localization, stability or altering their interactions with other protein molecules. The native 14-3-3 proteins are present in form of homo- and hetero-dimers. The most structurally variable N-and C-termini are responsible for isoform specific protein-protein interactions, and cellular localization. In plant cell, 14-3-3 proteins appear to play an important role in regulation of key enzymes of carbon and nitrogen metabolism, modulation ion pumps and channels. They are also involved in signal transduction pathways and even in gene expression.     

Deciphering the role of 14-3-3 proteins

14-3-3 Proteins are found to bind to a growing number of eukaryotic proteins and evidence is accumulating that 14-3-3 proteins serve as modulators of enzyme activity. Several 14-3-3 protein recognition motifs have been identified and an increasing number of target proteins have been found to contain more than one binding site for a 14-3-3 protein. It is thus possible that 14-3-3 dimers function as clamps that simultaneously bind to two motifs within a single binding partner. Phosphorylation of a number of binding motifs has been shown to increase the affinity for 14-3-3 proteins but other mechanisms also regulate the association. It has recently been demonstrated that fusicoccin induces a tight association between 14-3-3 proteins and the plant plasma membrane H⁺-ATPase. Phorbol esters and other hydrophobic molecules may have a similar effect on the association between 14-3-3 proteins and specific binding partners.     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.     

Depletion of 14-3-3 proteins in bloodstream-form Trypanosoma brucei inhibits variant surface glycoprotein recycling

Bloodstream-form Trypanosoma brucei have two 14-3-3 proteins, which are required for parasite multiplication. We here describe the effects of 14-3-3 depletion on vesicular transport of variant surface glycoprotein (VSG). 14-3-3 depletion had no detectable effect on de novo synthesis and trafficking of VSG to the cell surface, or on VSG endocytosis. Despite strong inhibition of cell division, the flagellar pocket was not enlarged and the ultrastructure of internal organelles appeared normal. The Rab11-positive recycling endosome compartment was, however, fivefold smaller than normal, and the rate of return of recycling VSG to the surface was correspondingly reduced. Down-regulating 14-3-3 also prevented enlargement of the flagellar pocket by clathrin depletion. These results suggest that there is a remarkably specific requirement for 14-3-3 in normal functioning of the Rab11-positive recycling endosome compartment.     

14-3-3 proteins: regulators of numerous eukaryotic proteins

14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.     

Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins

The conductance of the vacuolar membrane at elevated cytosolic Ca(2+) levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca(2+) concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca(2+)-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.     

Survival-promoting functions of 14-3-3 proteins

The 14-3-3 proteins are a family of phosphoserine/phosphothreonine-binding molecules that control the function of a wide array of cellular proteins. We suggest that one function of 14-3-3 is to support cell survival. 14-3-3 proteins promote survival in part by antagonizing the activity of associated proapoptotic proteins, including Bad and apoptosis signal-regulating kinase 1 (ASK1). Indeed, expression of 14-3-3 inhibitor peptides in cells is sufficient to induce apoptosis. Interestingly, these 14-3-3 antagonist peptides can sensitize cells for effective killing by anticancer agents such as cisplatin. Thus, 14-3-3 may be part of the cellular machinery that maintains cell survival, and targeting 14-3-3-ligand interactions may be a useful strategy to enhance the efficacy of conventional anticancer agents.     

14-3-3 proteins in neurological disorders

14-3-3 proteins were originally discovered as a family of proteins that are highly expressed in the brain. Through interactions with a multitude of binding partners, 14-3-3 proteins impact many aspects of brain function including neural signaling, neuronal development and neuroprotection. Although much remains to be learned and understood, 14-3-3 proteins have been implicated in a variety of neurological disorders based on evidence from both clinical and laboratory studies. Here we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins in both neurodegenerative and neuropsychiatric diseases.