The multistructural forms of box C/D ribonucleoprotein particles.

Structural biology studies of archaeal and yeast box C/D ribonucleoprotein particles (RNPs) reveal a surprisingly wide range of forms. If form ever follows function, the different structures of box C/D small ribonucleoprotein particles (snoRNPs) may reflect their versatile functional roles beyond what has been recognized. A large majority of box C/D RNPs serve to site-specifically methylate the ribosomal RNA, typically as independent complexes. Select members of the box C/D snoRNPs also are essential components of the megadalton RNP enzyme, the small subunit processome that is responsible for processing ribosomal RNA. Other box C/D RNPs continue to be uncovered with either unexpected or unknown functions. We summarize currently known box C/D RNP structures in this review and identify the Nop56/58 and box C/D RNA subunits as the key elements underlying the observed structural diversity, and likely, the diverse functional roles of box C/D RNPs.

Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.

Functional Interrogation of the N-Terminal Lid of MDMX in p53 Binding via Native Chemical Ligation.

The homologous proteins MDM2 and MDMX negatively regulate the tumor suppressor protein p53 by antagonizing p53 transactivation activity and targeting p53 for degradation. MDM2 and MDMX bind to p53 via N-terminal p53-binding domains to control the level of p53. The N-terminal regions of MDM2 and MDMX are modified in vivo under stressed conditions, suggesting that modifications to MDM2/MDMX also may affect the p53-MDM2/MDMX interaction. Ample evidence suggests that the MDM2 lid (residues 1-24) is partially structured and significantly reduces its binding affinity with p53 several fold. Since MDM2 and MDMX possess very similar p53-binding domains but different lids, however, the function of the N-terminal lid of MDMX still remains poorly understood. Using a native chemical ligation technique, the p53-binding domain of MDMX, (1-108)MDMX, and its N-terminal lid (residues 1-23) truncated analogue (24-108)MDMX were chemically synthesized. We comparatively characterized their structures by circular dichroism (CD) spectra, and measured their binding affinities with a panel of p53-derived peptide ligands by fluorescence polarization and surface plasmon resonance assays. Our results indicate that, as opposed to the lid of MDM2, the lid of MDMX has little effect on p53-binding, adopts no structural conformation, and has rare auto-inhibitory function. Different lid modifications of MDM2 and MDMX are functionally different with respect to p53 binding, which should be considered when designing dual specific inhibitors of MDM2 and MDMX.

Molecular sled sequences are common in mammalian proteins.

Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins.

Structural basis of how stress-induced MDMX phosphorylation activates p53.

The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage.

Redox state of p63 and p73 core domains regulates sequence-specific DNA binding.

Cysteine oxidation and covalent modification of redox sensitive transcription factors including p53 are known, among others, as important events in cell response to oxidative stress. All p53 family proteins p53, p63 and p73 act as stress-responsive transcription factors. Oxidation of p53 central DNA binding domain destroys its structure and abolishes its sequence-specific binding by affecting zinc ion coordination at the protein-DNA interface. Proteins p63 and p73 can bind the same response elements as p53 but exhibit distinct functions. Moreover, all three proteins contain highly conserved cysteines in central DNA binding domain suitable for possible redox modulation. In this work we report for the first time the redox sensitivity of p63 and p73 core domains to a thiol oxidizing agent azodicarboxylic acid bis[dimethylamide] (diamide). Oxidation of both p63 and p73 abolished sequence-specific binding to p53 consensus sequence, depending on the agent concentration. In the presence of specific DNA all p53 family core domains were partially protected against loss of DNA binding activity due to diamide treatment. Furthermore, we detected conditional reversibility of core domain oxidation for all p53 family members and a role of zinc ions in this process. We showed that p63 and p73 proteins had greater ability to resist the diamide oxidation in comparison with p53. Our results show p63 and p73 as redox sensitive proteins with possible functionality in response of p53 family proteins to oxidative stress.

Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms.

Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s(-1) (electrons per s)], UV-visible signatures of oxidized and reduced states and ability to form the P(M) ['peroxy' (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.

Inhibiting the nucleation of amyloid structure in a huntingtin fragment by targeting α-helix-rich oligomeric intermediates.

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with D-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.

Synthesis of pathological and nonpathological human exon 1 huntingtin.

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG-triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly-Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67-amino acid-long polypeptide plus a variable poly-Q stretch, is sufficient to cause full HD-like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109-amino acid-long exon 1 huntingtin peptide including a poly-Q stretch of 42 glutamines. This microwave-assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90-amino acid long including a poly-Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha-helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large-scale pre-screenings for aggregation inhibitors, further structural analyses as well as protein-protein interaction studies.

Association between anti-nucleophosmin and anti-cardiolipin antibodies in (NZW x BXSB)F1 mice and human systemic lupus erythematosus.

We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW x BXSB)F1 (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.

Vesicle membrane interactions of penetratin analogues.

Reports on serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides, also denoted protein transduction domains, have demonstrated the need for a reevaluation of the current understanding of peptide-mediated cellular delivery of large, hydrophilic molecules. In a recent study on the internalization in unfixed cells of penetratin and its analogues in which tryptophans are substituted for phenylalanines (Pen2W2F), lysines for arginines (PenArg), and arginines for lysines (PenLys), we revealed large dissimilarities in cell interactions among the peptides [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107]. We here investigated possible correlations with their respective affinities for the lipid membranes of large unilamellar vesicles. The variations found in membrane affinity correlated qualitatively with differences in hydrophobicity among the peptides but were by far too small to account for the striking differences in cell membrane binding. Interestingly, we found that the inclusion of a small fraction of lipids conjugated to poly(ethylene glycol) (PEG) in the vesicles both stabilized the vesicle dispersion against peptide-induced aggregation and, furthermore, enhanced the binding of the peptides to the membrane. By use of PEG-conjugated lipids, it could be shown that vesicle aggregation drives an alpha-helix to beta-sheet conformational transition for these peptides. A similar transition was discovered at submicellar concentrations of sodium dodecyl sulfate in aqueous solution for all peptides except PenLys. Finally, significant changes of the contributions to CD spectra from aromatic residues due to their insertion into the membrane were observed.

A photoregulated ligand for the nuclear import receptor karyopherin alpha.

The ability to orchestrate the transport of proteins between nucleus and cytoplasm provides cells with a powerful regulatory mechanism. Selective translocation between these compartments is often used to propagate cellular signals, and it is an intimate part of the processes that control cell division, viral replication, and other cellular events. Therefore, precise experimental control over protein localization, through the agency of light, would provide a powerful tool for the study and manipulation of these events. To this end, a prototype photoregulated nuclear localization signal (NLS) was derived from a native NLS. A library of 30 mutants of the bipartite NLS from Xenopus laevis nucleoplasmin containing a novel, photoisomerizable amino acid was prepared by parallel, solid-phase synthesis and screened in vitro for binding to the nuclear import receptor karyopherin alpha, which mediates the nuclear import of cellular proteins. A single peptide was identified in which the cis and trans photoisomers bind the receptor differentially. The strategy used to obtain this peptide is systematic and empirical; therefore, it is potentially applicable to any peptide-receptor system.

Characterization of the DNA binding properties of the bHLH domain of Deadpan to single and tandem sites.

The basic helix-loop-helix domain of the Drosophila transcription factor Deadpan (Dpn) was prepared by total chemical protein synthesis in order to characterize its DNA binding properties. Circular dichroism spectroscopy was used to correlate structural changes in Dpn with physiologically relevant monovalent (KCl) and divalent (MgCl2) cation concentrations. In addition, we have used electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy methods to determine equilibrium dissociation constants for the interaction of Dpn with two biologically relevant promoters involved in neural development and sex determination pathways. In this study, we have optimized DNA binding conditions for Dpn, and we have found a markedly higher DNA binding affinity for Dpn than reported for other bHLH domain transcription factors. Dpn binds as a homodimer (Kd = 2.6 nM) to double-stranded oligonucleotides containing the binding site CACGCG. In addition, we found that Dpn bound with the same affinity to a single or tandem binding site, indicating no cooperativity between adjacent DNA-bound Dpn dimers. DNA binding was also monitored as a function of physiologically relevant KCl and MgCl2 concentrations, and we found that this activity was significantly different in the presence and absence of the nonspecific competitor poly(dI-dC). Moreover, Dpn displayed moderate sequence selectivity, exhibiting a 100-fold higher binding affinity for specific DNA than for poly(dI-dC). This study constitutes the first detailed biophysical characterization of the DNA binding properties of a bHLH protein.

A novel nuclear import pathway for the transcription factor TFIIS.

We have identified a novel pathway for protein import into the nucleus. We have shown that the previously identified but uncharacterized yeast protein Nmd5p functions as a karyopherin. It was therefore designated Kap119p (karyopherin with Mr of 119 kD). We localized Kap119p to both the nucleus and the cytoplasm. We identified the transcription elongation factor TFIIS as its major cognate import substrate. The cytoplasmic Kap119p exists as an approximately stoichiometric complex with TFIIS. RanGTP, not RanGDP, dissociated the isolated Kap119p/TFIIS complex and bound to Kap119p. Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays. In wild-type cells, TFIIS was primarily localized to the nucleus. In a strain where KAP119 has been deleted, TFIIS was mislocalized to the cytoplasm indicating that TFIIS is imported into the nucleus by Kap119p. The transport of various substrates that use other karyopherin-mediated import or export pathways was not affected in a kap119Delta strain. Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro.

Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0 degree C and is Ca(2+)-independent. By contrast, translocation requires ATP hydrolysis, Ca2+, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells.

Nuclear transport in vitro.

In this paper, progress towards the goal of understanding communication between the nucleus and cytoplasm using an in vitro system is reviewed. To probe the mechanism of nuclear targeting, we developed an in vitro transport system and have begun to dissect the highly selective process of nuclear transport. The basic parameters of transport were defined using an easily isolated nuclear protein, nucleoplasmin. To study the interaction of nuclear targeting signals with the pore, an artificial nuclear transport substrate was constructed, which consists of human serum albumin coupled to the signal sequence of the SV40 T-antigen. A similar peptide-protein conjugate was made using a mutant signal sequence. These conjugates were fluorescently labeled and/or tagged with gold and tested for transport in the in vitro system. High levels of nuclear transport of the wild-type signal sequence-containing protein were observed, while no transport of the mutant signal sequence-containing protein was seen. Thus, the in vitro system correctly recognizes the single amino acid change between the wild-type and mutant signal sequences. We found that the observed nuclear transport was completely dependent on the presence of ATP. Using the in vitro system we identified a specific inhibitor of nuclear transport, the lectin wheat germ agglutinin (WGA), which we find binds directly to the nuclear pore. Probing blots of nuclear proteins with 125I-WGA identified a family of nuclear pore glycoproteins, including one major glycoprotein of 62K (K = 10(3)Mr) molecular weight. With the inhibitor and the in vitro assay, it has been possible to experimentally separate nuclear transport into two steps: (1) a step in which the signal sequence-bearing protein binds to the pore, followed by (2) a step in which the protein translocates through the pore. It is this second step which is the ATP-dependent step of transport, since pore binding but not translocation was seen to occur in the absence of ATP.