5 '-Untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5'-untranslated regions (5'-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, similar to10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.

Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Role of oxidative stress in age-associated chronic kidney pathologies

The kidneys exhibit age-associated deterioration in function via a loss of 20% to 25% kidney mass, particularly from the renal cortex and increased fibrosis. Oxidative stress has been found to mediate age-associated renal cell injury and cell death, particularly apoptosis. Oxidative stress results from an imbalance between the levels of free radicals generated during aerobic metabolism, inflammation, and infection and the safe breakdown of these species by endogenous and exogenous scavengers. Other factors may influence these pathologies. For example, growth hormone and caloric restriction have been shown to influence life span, although neither method of prolonging life is likely to find general acceptance in humans. Some genetic knockout models offer promise; for example, knockout of the p66 isoform of the Shc gene in mice increases life span by 30%, but appetite, size, and fertility are retained. Whether the increase in life span is via increased kidney health is not yet clear, but decreasing the age-related renal pathologies will no doubt aid in increasing life span and health in general. This review looks at the role and modulation of factors that influence life span, in particular modulation of oxidative stress, with particular relevance to age-related renal pathologies. (C) 2005 by the National Kidney Foundation, Inc.

Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines

There is evidence to suggest that plasma membrane Ca2+-ATPase (PMCA) isoforms are important mediators of mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184135 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study. (c) 2005 Elsevier Inc. All rights reserved.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier Ltd. All rights reserved.

Developmental expression of two Haliotis asinina hemocyanin isoforms

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

PKC-alpha-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Go-6976 and 2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10-20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

Silencing of integrated human papillomavirus type 18 oncogene transcription in cells expressing SerpinB2

The serine protease inhibitor SerpinB2 (PAI-2), a major product of differentiating squamous epithelial cells, has recently been shown to bind and protect the retinoblastoma protein (Rb) from degradation. In human papillomavirus type 18 (HPV-18) -transformed epithelial cells the expression of the E6 and E7 oncoproteins is controlled by the HPV-18 upstream regulatory region (URR). Here we illustrate that PAI-2 expression in the HPV-18-transformed cervical carcinoma line HeLa resulted in the restoration of Rb expression, which led to the functional silencing of transcription from the HPV-18 URR. This caused loss of E7 protein expression and restoration of multiple E6- and E7-targeted host proteins, including p53, c-Myc, and c-Jun. Rb expression emerged as sufficient for the transcriptional repression of the URR, with repression mediated via the C/EB beta-YY1 binding site (URR 7709 to 7719). In contrast to HeLa cells, where the C/EBP beta-YY1 dimer binds this site, in PAI-2- and/or Rb-expressing cells the site was occupied by the dominant-negative C/EBP beta isoform liver-enriched transcriptional inhibitory protein (LIP). PAI-2 expression thus has a potent suppressive effect on HPV-18 oncogene transcription mediated by Rb and LIP, a finding with potential implications for prognosis and treatment of HPV-transformed lesions.

The human stress-activated protein kinase-interacting 1 gene encodes JNK-binding proteins

The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. In this study, we have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin 1 alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin 1 polyclonal antibody showed that full-length Sin I and several smaller isoforms are widely expressed. Sin 1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1 alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1 alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK. (c) 2004 Elsevier Inc. All rights reserved.

FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B I) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear-import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds - Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney ( OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

Differential use of signal peptides and membrane domains is a common occurrence in the protein output of transcriptional units

Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi- spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units ( TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our highconfidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome.