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Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1–HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

The ATR–dependent DNA damage checkpoint is one of the major checkpoint pathways. The interaction between the Rad17–RFC2-5 and 9–1–1 complexes is central to the ATR–Chk1 pathway. However, little is known about the regulation of the interaction. We recently showed that vertebrate Rad17 proteins share a conserved C-terminal tail and that the C-terminal tails have a conserved amino acid motif named iVERGE that must be intact for the interaction between Rad17 and the 9‒1‒1 complex. In human Rad17, the Y665 and S667 residues are conserved in iVERGE. The Rad17-S667 residue is phosphorylated by CK2, and the phosphorylation is important for the interaction with the 9‒1‒1 complex. Here, we show that a C-terminal threonine residue of Rad17, T670 in human Rad17, is constitutively phosphorylated in vivo. The T670 phosphorylation is important for the S667 phosphorylation, and vice versa. Phosphomimetic mutations in the T670 residue promote the interaction with the 9–1–1 complex. The T670 and Y665 residues show functional redundancy, and their roles are dependent on the S667 phosphorylation. Rad17-T670 is phosphorylated by casein kinase 1δ/e. Our data suggest that iVERGE integrates multiple signaling pathways to regulate the ATR–Chk1 pathway.

Darwin’s bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline’s toughness, enabling giant webs. Caerostris darwini’s MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin’s bark spider, presenting innovative designs for engineering biomaterials., Jessica Garb et al. report the transcriptome of the major ampullate silk gland of Darwin’s bark spider, known for its extraordinarily tough silk. They identify a novel predicted protein, MaSp4, which is highly divergent compared to related proteins in other species and has properties predicted to confer silk extensibility.

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

Background: We discovered that lipopolysaccharide (LPS, an initiating factor in sepsis) and hypoxia-reoxygenation (H/R, a confounding factor) reduce electrical coupling between microvascular endothelial cells from wild-type (WT) but not Cx40-/- mice. Because Cx40 knockout could result in nonspecific effects, this discovery may not establish the causal relationship between Cx40 and reduced coupling. Using the same cell culture model, we aimed to address this uncertainty by using the rescue-of-function approach. Methods/Results: Electrical coupling between endothelial cells (hind-limb muscle origin) was determined by electrophysiology. LPS, H/R and concurrent LPS + H/R reduced coupling between WT but not Cx40-/- cells. The defect in Cx40-/- cells was rescued by ectopic expression of Cx40, after infecting the cells with adenovirus encoding Cx40. Cx40-/- cells were also engineered to express mutant Cx40 that lacked the carboxyl terminal domain beginning at residue 236 (Cx40Δ237-358) or 344 (Cx40Δ345-358). No response to inflammatory stimuli was observed in cells expressing either of these 2 mutants. Conclusion: Our data establish the causal relationship between Cx40 and reduced coupling and suggest that the 345-358 amino acid motif of the Cx40 carboxyl terminal is required for reduced coupling. Cx40 may participate in compromised conducted response in the microvasculature during sepsis.

Emerging evidence supports the role for the intracellular domains of amyloid precursor protein (APP) in the physiology and function of APP. In this short report, I discuss the hypothesis that mutation of Tyr682 on the Y682 ENPTY687 C-terminal motif of APP may be directly or indirectly associated with alterations in APP functioning and activity, leading to neuronal defects and deficits. Mutation of Tyr682 induces an early and progressive age-dependent cognitive and locomotor decline that is associated with a loss of synaptic connections, a decrease in cholinergic tone, and defects in NGF signaling. These findings support a model in which APP-C-terminal domain exerts a pathogenic function in neuronal development and decline, and suggest that Tyr682 potentially could modulate the properties of APP metabolites in humans.

Avian influenza viruses in wild birds at the Jeziorsko reservoir in Poland in 2008-2010 During a 3-year surveillance study for avian influenza virus (AIV) infections at the Jeziorsko reservoir in central Poland, 549 oropharyngeal or cloacal swabs from 366 birds of 14 species belonging to 3 orders (Anseriformes, Charadriiformes and Gruiformes) were tested. AIV was detected in 14 birds (3.8%): Common Teals (12x), Mallard (1x) and Garganey (1x). Three potentially dangerous H5 AIV were detected in Common Teals (2x) and Garganey (1x) but all of them revealed a low pathogenic pathotype. A unique cleavage site amino acid motif PQREIR*GLF was found in one H5 isolate from a Garganey.

Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins, leading to their degradation and the liberation of nuclear factor κB for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor, at a resolution of 3.6 A. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix–loop–helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity and instead is required for IKKβ activation. Other IKK family members, IKKα, TBK1 and IKK-i, may have a similar trimodular architecture and function. IKK (inhibitor of κB kinase) is an important element in the activation of nuclear factor κB (NF-κB) transcription factors — master regulators of inflammatory, immune and apoptotic responses. Here Wu and colleagues describe the long awaited X-ray structure of a member of the IKK family in combination with an inhibitor. They show that IKKβ has a tri-molecular architecture that contains the kinase domain, a ubiquitin-like domain and an extended scaffolding and dimerization domain, and discuss the functional implications.

In 2002-2003, velogenic Newcastle Disease Virus outbreaks, closely related to the Mexican isolates, were confirmed in the United States (U.S.) in southern California, Arizona, Nevada, and Texas. In this report, virulent NDVs isolated in Mexico between 1998 and 2006 were subjected to biologic characterization, using standard pathogenicity tests, and to phylogenetic analysis. Chicken embryo mean death time (MDT) test results ranged from 39.7 to 61.5 hours, and intracerebral pathogenicity index (ICPI) values were between 1.59 and 1.94, compared to a possible maximum value of 2.0. These isolates showed a dibasic amino acid motif at the fusion protein cleavage site sequence required for host systemic replication. Phylogenetic analysis indicated that the Mexican virulent NDVs belong to the class II, genotype V viruses and can be clearly divided in two groups as follows: isolates from 1998 to 2001 with close epidemiologic relationship with the latest U.S. NDV outbreaks, and phylogenetically distinct viruses, isolated from 2004 to 2006, which showed higher virulence. The assessment of the evolution of viruses from Mexico and other neighboring countries will aid in the U.S surveillance efforts for early detection of highly virulent NDV.

The elasticity of spider MAA silks containing varying proline content was investigated and compared with that of silkworm ( Bombyx mori) silk. For silks with similar breaking strain (suggesting similar molecular order), the elasticity appears to increase with increasing proline content. Particularly, across all spider silks, intra- and interspecies relationships are found between capacity to shrink (Csh) and strain recovery, while only the interspecies relationship is found between Csh and work recovery. Four factors, that is, molecular orientation, crystallinity, amino acid motif, and hydration, are discussed to explain the origin of silk's elasticity. Our study corroborates the view that proline-containing motifs contribute to the elasticity of not only spider silks, but also other bioelastomers.

Background The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. Scope of review p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. Major conclusions The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. General significance The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets.

Item does not contain fulltext Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, with an approximate incidence of 1 in 3700 individuals worldwide. Mutations in the retinitis pigmentosa 1 (RP1) gene are responsible for about 5-10% cases of autosomal dominant RP. The RP1 gene is specifically expressed in the photoreceptor layers of the postnatal retina and encodes a predicted protein characterised by the presence of two doublecortin (DC) domains, known to be implicated in microtubule binding. We identified and characterised, both in human and in mouse, a novel mammalian gene, termed Retinitis Pigmentosa1-like1 (RP1L1), because of its significant sequence similarity to the RP1 gene product. The sequence homology between RP1 and RP1L1 was found to be mostly restricted to the DC domains and to the N-terminal region, including the first 350 amino acids. The RP1L1 gene was also found to be conserved in distant vertebrates, since we identified a homologue in Fugu rubripes (pufferfish). Similar to RP1, RP1L1 expression is restricted to the postnatal retina, as determined by semiquantitative reverse transcriptase-PCR and Northern analysis. The retina-specific expression and the sequence similarity to RP1 render RP1L1 a potential candidate for inherited retinal disorders.

SummaryTo successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits.

Fruit fly species of the genus Drosophila show a remarkable variation in sperm length. Some of them produce gigantic sperm several times the total male body length. Sperm of Drosophila hydei, for example, are more than 20 mm long. Little is known about the advantage of such elongated sperm or about the proteins that stabilize their thin flagellar tails. Recently, two members of a novel gene family Dhmst101(1) and Dhmst101(2), whose gene products are associated with the sperm tail, were isolated and characterized. Here a third member of this gene family, Dhmst101(3), is described. It was previously demonstrated that all three genes are located in a single small cluster on chromosome 5 of D. hydei. They are located within 15 kb of genomic DNA, oriented in the same direction and transcribed testis-specifically. The encoded sperm tail-specific proteins are mainly composed of tandemly arranged repeats of a highly charged, cysteine-containing motif of 16 amino acids with the consensus sequence KKKCA/EEAAKKEKEAAE. Experiments with synthetic repeat monomers and dimers have demonstrated a tendency for alpha-helical rod formation, which increased strongly with an increase in repeat number. Therefore, Dhmst101 proteins with 7-60 repeats with regularly spaced cystein-residues are thus expected to form long alpha-helical rods cross-linked by numerous Cys-Cys bridges. Here we apply immunoelectron microscopy and monospecific antibodies, alpha-mst101, raised against the KKKCAEAAKKEKEAAE-motif to investigate the distribution of Dhmst101 proteins within the sperm tail of D. hydei. We show that Dhmst101 proteins are part of the outer sheath of the sperm tail where they presumably help to provide a tight but elastic envelope for the extremely extended spermatozoa of D. hydei.

Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC− strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC− strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA’s von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics., IMPORTANCE Catheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens.

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3–CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses., Author Summary Nipah (NiV) and Hendra (HeV) viruses are two lethal emerging zoonotic paramyxoviruses. In addition to acute infection, these viruses may lead to late-onset disease or relapse of encephalitis years after initial infection, as well as persistent or delayed neurological sequelae. We present a new strategy to prevent and treat NiV/HeV infection that may be broadly applicable for enveloped viral pathogens. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection, and blocking this step can prevent or treat infection, as clinically validated for HIV. For paramyxoviruses, however, peptides that bind the viral fusion protein have been shown to inhibit fusion in vitro, but not in vivo. The new strategy that we present here opens the door to clinically effective paramyxovirus fusion-inhibitory peptides. By targeting fusion-inhibitory peptides to the target membrane using a cholesterol tag, we capture an early stage in the viral fusion-activation process, thus drastically enhancing the efficacy of these peptides at inhibiting viral entry. Importantly, this strategy prevents and treats lethal Nipah virus infection in vivo. Membrane targeting of antiviral peptides thus offers a new approach to development of highly effective peptide fusion antivirals against important human pathogens.

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright © 2008 John Wiley & Sons, Ltd.

Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide-tagged cargoes, although some in vitro studies have suggested the relevance of active processes in Tat peptide-driven nuclear translocation. These issues are addressed by comparing Tat peptide-induced transport properties with well-established passive diffusion and active import benchmarks in living cells. Specifically, we examine several constructs of increasing molecular weight (MW) both below and above the threshold for passive diffusion through the nuclear pore. The resulting sub-cellular localization is analyzed by confocal imaging, and construct intracellular dynamics is investigated by fluorescence recovery after photobleaching (FRAP) real-time imaging. Our experiments yield the characteristic transport parameters of Tat peptide intra-cytoplasm dynamics and nucleus/cytoplasm shuttling. These results allow us to elucidate the mechanism of Tat peptide-driven nuclear permeation, demonstrating that it crosses the nuclear envelope (NE) by passive diffusion. Finally, we discuss the limitations of this route in terms of acceptable cargo size.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

Myf5 is a nuclear protein and one of the basic helix-loop-helix (bHLH) myogenic factors that play an important role in muscle specification and differentiation. The motif responsible for the nuclear translocation of Myf5 was unknown. Using on-line monitoring of EGFP (enhanced green fluorescent protein)-tagged zebrafish Myf5 translocation, we demonstrated that Myf5-EGFP protein resided in the nucleoplasm and nucleolus of zebrafish fibroblast cell lines (ZEM2S and ZF4), mammalian nonmuscle cell line (COS1), and muscle cell lines (RD and C2C12). In contrast, zebrafish MyoD-EGFP was localized in the nucleus but did not condense in the nucleolus. Using indirect immunofluorescent staining, we determined that zebrafish Myf5 was colocalized with nucleophosmin/B23, a nucleolus protein. Deletion analysis revealed that amino acid residues 60 to 82 (60KRKASTVDRRRAATMRERRRLKK82) of Myf5 were sufficient and necessary for nucleolus targeting. A GST pulldown assay followed by Western analysis showed that nucleolin/C23 could be pulled down specifically by GST-Myf5, but not by GST-MyoD. Based on these findings, we propose that the distinct functions of Myf5 and MyoD may result from their differential binding affinity to nucleolin/C23.

Na v 1.5, the cardiac isoform of the voltage-gated Na + channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na v 1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na v 1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na v 1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na v 1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Na v 1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na v 1.5 and Nedd4-2. Na v 1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na v 1.5. Consistent with the functional findings, localization at the cell periphery of Na v 1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na v 1.5, suggesting that Nedd4-2 is a specific regulator of Na v 1.5. These results demonstrate that Na v 1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na v 1.5 by decreasing the channel density at the cell surface.

Here we identify the determinants of the nucleotide-binding ability associated with the P-loop-containing proteins, inferring their functional importance from their structural convergence to a unique three-dimensional (3D) motif. (1) A new surface 3D pattern is identified for the P-loop nucleotide-binding region, which is more selective than the corresponding sequence pattern; (2) the signature displays one residue that we propose is the determinant for the guanine-binding ability (the residues aligned to ras D119; this residue is known to be important only in the G-proteins, we extend the prediction to all the other P-loop-containing proteins); and (3) two cases of convergent evolution at the molecular level are highlighted in the analysis of the active site: the positive charge aligned to ras K117 and the arginine residues aligned to the GAP arginine finger. The analysis of the residues conserved on protein surfaces allows one to identify new functional or evolutionary relationships among protein structures that would not be detectable by conventional sequence or structure comparison methods. (C) 2000 Academic Press.

Recombinant human growth hormone (rhGH) is now used routinely in the treatment of congenital and acquired GH deficiency (GHD). Additional indications for long-term administration of rhGH such as osteoporosis and aging have been proposed, but concerns regarding the safety of such prolonged courses of rhGH have been raised. Administration of rhGH is associated with glucose intolerance, higher plasma insulin concentrations and fluid retention. To add to these potential problems is the finding that rhGH may increase plasma concentrations of the atherogenic lipoprotein(a) (Lp(a)) particle (1, 2). In Caucasians, increased plasma concentrations of Lp(a) are associated with an increased risk of cardiovascular disease (3), but the mechanism by which Lp(a) is atherogenic has not been fully elucidated. Lp(a) is present only in hedgehogs, great apes and humans (4). In humans, plasma concentrations of Lp(a) vary over a 1000-fold range between individuals. Plasma Lp(a) concentrations are primarily determined by sequences at or close to the gene encoding apolipoprotein(a) (apo(a)), a highly polymorphic glycoprotein that is covalently attached to apolipoprotein B-100 (apoB100) of low-density lipoprotein (LDL) in Lp(a) (5). apo(a) contains variable numbers of a tandemly repeated ,114 amino acid motif called kringle-4. Overall, there is an inverse relationship between the number of kringle-4 repeats encoded by the apo(a) gene and the associated plasma Lp(a) concentration (6, 7). Only a restricted number of physiological, pharmacological or environmental factors influence plasma Lp(a) concentrations (8); GH is one of these factors. Over the past 5 years, more than 20 studies, including six placebo-controlled double-blind studies, have examined the effect of the administration of rhGH on plasma Lp(a) concentrations. Most studies show that rhGH administration is associated with a 110–200% increase in plasma Lp(a) concentrations, but in two studies, plasma Lp(a) concentrations remained unchanged after 12 months of rhGH therapy (9, 10). Both of these studies were performed in Japanese children, which may reflect inter-ethnic differences in the effect of rhGH on plasma Lp(a) concentrations. In this issue of European Journal of Endocrinology, Nolte et al. (11) report the effect of rhGH administration on plasma Lp(a) concentrations in a randomised, placebo-controlled, double-blind 12-month study involving 32 adults with GHD. The authors found that rhGH administration induced an ,140% increase in plasma Lp(a) concentrations and an ,14% decrease in LDL-cholesterol concentrations. This well-designed and well-executed study demonstrates once again that administration of rhGH to GHD adults is associated with a significant increase in plasma Lp(a) concentrations. Should we, as clinicians, be concerned about the Lp(a)-increasing effect of rhGH? The answer to this question is not as straightforward as one might suppose. The increase in plasma Lp(a) concentrations associated with rhGH substitution in GHD patients is modest, especially in relationship to the wide inter-individual variability in plasma Lp(a) concentrations in the general population. A direct relationship exists between plasma LDL-cholesterol concentrations and the incidence of coronary atherosclerosis. In contrast, the atherogenicity of Lp(a) does not appear to be concentrationdependent over the entire range of plasma Lp(a) concentrations. Lp(a) is associated with a significant risk when plasma Lp(a) concentrations are greater than about 20–30 mg/dl (3). If subjects in the study by Nolte’s group were classified as having low Lp(a) concentrations (i.e. 20 mg/dl), only two of 32 subjects treated with rhGH moved from the lowto the high-risk category with treatment. The Lp(a)-increasing effect of rhGH is coupled with a significant reduction in the plasma LDL-cholesterol concentrations (1, 2, 11). GHD patients tend to have higher plasma LDL-cholesterol concentrations than healthy controls and are at a significantly greater risk of developing cardiovascular events (12), despite their lower plasma Lp(a) concentrations. The beneficial LDL-decreasing effect of rhGH in GHD individuals may counterbalance the potentially detrimental effect of the increase in plasma Lp(a) concentrations. Moreover, in non-GHD subjects, reduction of plasma LDL-cholesterol concentrations using pharmacological agents that do not affect plasma Lp(a) significantly reduces coronary risk (13). Even in individuals with high plasma concentrations of Lp(a), it appears that decreasing plasma LDL-cholesterol concentrations may obviate the atherogenic effects of a high plasma Lp(a) concentration (14). European Journal of Endocrinology (1997) 137 450–452 ISSN 0804-4643