Exploring the Sea Urchin Neuropeptide Landscape by Mass Spectrometry.

Neuropeptides are essential cell-to-cell signaling messengers and serve important regulatory roles in animals. Although remarkable progress has been made in peptide identification across the Metazoa, for some phyla such as Echinodermata, limited neuropeptides are known and even fewer have been verified on the protein level. We employed peptidomic approaches using bioinformatics and mass spectrometry (MS) to experimentally confirm 23 prohormones and to characterize a new prohormone in nervous system tissue from Strongylocentrotus purpuratus, the purple sea urchin. Ninety-three distinct peptides from known and novel prohormones were detected with MS from extracts of the radial nerves, many of which are reported or experimentally confirmed here for the first time, representing a large-scale study of neuropeptides from the phylum Echinodermata. Many of the identified peptides and their precursor proteins have low homology to known prohormones from other species/phyla and are unique to the sea urchin. By pairing bioinformatics with MS, the capacity to characterize novel peptides and annotate prohormone genes is enhanced. Graphical Abstract.

Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability.

We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.

Capturing Structural Heterogeneity in Chromatin Fibers.

Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome-nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome-nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells.

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

X-ray structure of the MMTV-A nucleosome core.

The conformation of DNA bound in nucleosomes depends on the DNA sequence. Questions such as how nucleosomes are positioned and how they potentially bind sequence-dependent nuclear factors require near-atomic resolution structures of the nucleosome core containing different DNA sequences; despite this, only the DNA for two similar α-satellite sequences and a sequence (601) selected in vitro have been visualized bound in the nucleosome core. Here we report the 2.6-Å resolution X-ray structure of a nucleosome core particle containing the DNA sequence of nucleosome A of the 3'-LTR of the mouse mammary tumor virus (147 bp MMTV-A). To our knowledge, this is the first nucleosome core particle structure containing a promoter sequence and crystallized from Mg(2+) ions. It reveals sequence-dependent DNA conformations not seen previously, including kinking into the DNA major groove.

Human anti-nucleolin recombinant immunoagent for cancer therapy.

Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies.

The metallo-ß-lactamases (MßLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in ß-lactam-containing antibiotics, and render the antibiotics inactive. All known MßLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MßL L1 with spin labels at positions 163 and 286 and analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was "diluted" by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

Structure of the full-length yeast Arp7-Arp9 heterodimer.

The nuclear actin-related proteins Arp7 and Arp9 are components of the yeast SWI/SNF and RSC chromatin-remodelling complexes. The 3.1 Šresolution crystal structure reported here shows that the full-length Arp7 and Arp9 proteins exist as a dimer without a requirement for additional polypeptides. Of the 11 actin-related proteins, Arp7 and Arp9 are the only two directly demonstrated to form a dimer within this family. The Arp7-Arp9 heterodimer is unlikely to form an actin-like filament based on modelling using the structure. The Arp7-Arp9 structure reveals that its dimerization interface is not altered when bound in a complex with the SWI/SNF Snf2 HSA domain and the regulatory protein Rtt102.

Urotensin II in invertebrates: from structure to function in Aplysia californica.

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.

Mechanistic and spectroscopic studies of metallo-ß-lactamase NDM-1.

In an effort to biochemically characterize metallo-ß-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP-NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a k(cat) of 16 s(-1) and a K(m) of 1.1 µM when using nitrocefin as a substrate, bound 2 equiv of Zn(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-ß-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-ß-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-ß-lactamase CcrA.

Nucleosome recognition and spacing by chromatin remodelling factor ISW1a.

Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.

Structural and kinetic studies on metallo-ß-lactamase IMP-1.

In an effort to probe for metal binding to metallo-ß-lactamase (MßL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.

Structure and mechanism of the chromatin remodelling factor ISW1a.

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.

Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

Automated unrestricted multigene recombineering for multiprotein complex production.

Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.

Quantitative peptidomics reveal brain peptide signatures of behavior.

The honey bee genome predicts approximately 100 peptides from 36 prohormones, but the functions of many of these peptides are unknown. We used differential isotope labeling combined with mass spectrometric analysis to quantify approximately 50% of known bee brain peptides in the context of foraging, with 8 showing robust and dynamic regulation. Some showed differences in brain abundance as a function of experience; specifically, nectar and pollen collection led to quick changes in abundance. These changes were related to the act of food collection, not ingestion, because foragers bring food back to the hive for storage rather than eating it themselves. Other peptide differences in brain abundance were seen in bees that either flew to a nectar feeder or a pollen feeder, but did not yet collect any food. These differences likely reflect well-known predispositions of some bees to collect either nectar or pollen, but not both. Tachykinin, PBAN, and sNPF were among the peptides with the strongest changes in association with nectar and pollen foraging. These peptides are known to be involved in regulating food intake in solitary insects, suggesting an evolutionary connection between that behavior and social foraging. These results demonstrate that it is now possible to use quantitative peptidomics to help determine which brain peptides are bioactive and to elucidate their function in the regulation of behavior.

MultiBac: multigene baculovirus-based eukaryotic protein complex production.

Multiprotein complexes are an emerging focus in current biology, resulting in a demand for advanced heterologous expression systems. This unit provides protocols for the expression of eukaryotic multiprotein complexes using multigene expression vectors. Homologous and site-specific recombinases facilitate their assembly. Thus, modification of individual subunits for revised expression studies is achieved with comparative ease. The strategy outlined here employs the MultiBac baculoviral expression system for multiprotein complexes as an example. Baculoviral expression does not require particular safety precautions due to the replication incompetence of baculovirus in mammalian hosts. The MultiBac system provides for improved protein production due to deletion of specific viral genes (V-cath, chiA). Most of the steps described in this unit are tailored for high-throughput approaches. The general strategy of rapidly combining encoding DNAs by recombination into multigene expression vectors for protein complex expression can also be applied to other prokaryotic or mammalian expression systems.

Projected [(1)H, (15)N]-HMQC-[ (1)H, (1)H]-NOESY for large molecular systems: application to a 121 kDa protein-DNA complex.

We present a projected [(1)H,(15)N]-HMQC-[(1)H,(1)H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate (15)N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation peaks also in spectra with low resolution by fully exploiting both (1)H and (15)N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of the experiment is not dependent on the projection angle for projections up to 45 degrees and no additional pulses or delays are required as compared to the conventional 2D [(1)H,(15)N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections, respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection experiment for the observation of an NOE interaction between two sequential glycines with degenerate (15)N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA.

Multiprotein expression strategy for structural biology of eukaryotic complexes.

The concept of the cell as a collection of multisubunit protein machines is emerging as a cornerstone of modern biology, and molecular-level study of these machines in most cases will require recombinant production. Here, we present and validate a strategy to rapidly produce, permutate, and posttranslationally modify large, eukaryotic multiprotein complexes by using DNA recombination in a process that is fully automatable. Parallel production of 12 protein complex variants within a period of weeks resulted in specimens of sufficient quantity and homogeneity for structural biology applications.

DNA stretching and extreme kinking in the nucleosome core.

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55 degrees kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.