A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.

The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

The association between family dynamics and Positive Youth Development in secondary education students.

INTRODUCTION: The Positive Youth Development (PYD) framework is a strengths-based approach to adolescence that states that adolescents will thrive if nurtured by the right developmental assets. The family is one of the most important developmental assets, but studies about the relationship between family dynamics and the overall PYD of adolescents are scarce. OBJECTIVE: The present study aims to examine the associations between five family dynamics indicators and PYD, while taking into account the role of gender. METHODS: A cross-sectional study was carried out with a representative sample of adolescents from the city of Huelva, Spain (n = 1,036). Data were collected in 14 randomly selected secondary education schools. A Structural Equation Model (SEM) was tested to determine the effect of family dynamics on PYD, both for the whole sample and within each gender. RESULTS: The SEM for the whole sample showed a positive effect of the family dynamics factor on the PYD factor, explaining 51.8% of its variance. The indicators of satisfaction with the relationship with the mother, satisfaction with the relationship with the father, frequency of engaging in joint family activities on weekends, and frequency of sharing daily occurrences at home showed factor loadings over 0.50, while the indicator of frequency of contribution to household chores had the lowest loading. The family dynamics factor in the model with the subsample of girls explained 54.8% of the variance in PYD, while in the SEM with the subsample of boys this factor explained 47.6% of it. Additionally, among girls, the relative influence of satisfaction in the relationship with the parents, as well as of frequently discussing the day at home, is higher than among boys. CONCLUSION: These results highlight a strong association between family dynamics indicators and PYD among adolescents and indicate that this relationship is stronger for girls than for boys. Intersectoral policies enhancing improvements in family dynamics (e.g., facilitating the practice of joint family activities on weekends) may have a relevant impact on PYD.

Phenotypic selection on an ornamental trait is not modulated by breeding density in a pied flycatcher population.

Most studies of phenotypic selection in the wild have focussed on morphological and life-history traits and looked at abiotic (climatic) variation as the main driver of selection. Consequently, our knowledge of the effects of biotic environmental variation on phenotypic selection on sexual traits is scarce. Population density can be considered a proxy for the intensity of intrasexual and intersexual competition and could therefore be a key factor influencing the covariation between individual fitness and the expression of sexual traits. Here, we used an individual-based data set from a population of pied flycatchers (Ficedula hypoleuca) monitored over 24 years to analyze the effect of breeding density on phenotypic selection on dorsal plumage colouration, a heritable and sexually selected ornament in males of this species. Using the number of recruits as a fitness proxy, our results showed overall stabilizing selection on male dorsal colouration, with intermediate phenotypes being favoured over extremely dark and dull individuals. However, our results did not support the hypothesis that breeding density mediates phenotypic selection on this sexual trait. We discuss the possible role of other biotic factors influencing selection on ornamental plumage.

Assessment of dissolved mercury by diffusive gradients in thin films devices in abandoned ponds impacted by small scale gold mining.

In order to fulfil the Minamata Convention on Mercury, it is necessary to monitor the Hg contamination in freshwater ecosystems nearby artisanal and small scale gold mining (ASGM) areas. Since most of these ASGM communities are located in remote areas, a convenient method for sampling, preserving and transporting samples is needed. In this study we evaluated the feasibility of the diffusive gradient in thin-films (DGT) technique to detect and quantify the labile fraction of Hg and other metals (Pb, Cu, Zn, Cd, Ni, Mn and Cr) in a hard-to-reach gold mining district in the state of Chocó, Colombia. We deployed DGT at sampling sites along the Atrato river and abandoned mining ponds (AMPs) which were deserted in different periods since 1997 to 2019 (6-15 years). In average, the labile THg concentrations in AMPs (148.9 ± 43.2 ng L-1) were a 50% higher than in the river water (99.9 ± 37.4 ng L-1). In the ponds, no significant differences were found in labile Hg with respect abandonment period. Labile Ni (0.9-493.1), Mn (1.33-11.48), Cu (0.030-2.233), and Zn (0.67-10.29) (in µg L-1) were found in higher amounts than for the rest of metals. Labile concentrations of metals are related with their downstream proximity to gold mining activities, being higher in devices deployed close to ASGM sites. Moreover, this study demonstrates the feasibility of the DGT technique to sample, transport, storage, and preserve labile Hg from hard-to-reach ASGM areas.

A human health risk assessment of methylmercury, arsenic and metals in a tropical river basin impacted by gold mining in the Colombian Pacific region.

The Atrato River basin is one of the most biodiverse areas worldwide, and paradoxically, it is one of the sites in Colombia with the highest environmental impact from gold mining. This study assessed the distribution of Hg, As, Pb, and Cd in 47 fish species (n = 1372) and the accumulative human health risk in inhabitants (n = 2325) from 13 municipalities located along the Atrato River basin. The results revealed that Hg and As in fish present a high potential human health risk based on their mean concentrations. Estimated daily intake (EDI) calculations showed that humans could present detrimental health effects, while that target hazard quotient (THQ) above 1 showed that the exposed population might experience noncarcinogenic health risks, mainly from the accumulative effects of Hg (80.4%) and As (18.2%). The species that would most affect the health of the inhabitants are carnivorous H. malabaricus, A. pardalis, P. schultzi, R. quelen, and C. kraussii, which are among the fourteen most consumed in the region. These species had values of estimated weekly intake (EWI) above the provisional tolerable weekly intake thresholds for MeHg (PTWI of 1.6 and 3.2 µg/kg bw/week for adults and children, respectively) in 7 of the 13 localities evaluated. According to the surveys, the calculated weekly allowable fish amount (MFW) showed that carnivorous fish may generate adverse effects on the consumers because the allowed MeHg is about 2 times higher than the upper reference limit. Other results indicate a significant carcinogenic health risk, mainly from As, in 8 of the 13 localities evaluated. Due to the high rates of unsatisfied basic needs and the monetary poverty in the region, the possibility that inhabitants can replace fish as the principal source of protein is low. Therefore, a food guidance is required to avoid risks, obtain nutritional benefits, and sustain fish populations.

Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant.

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

Fluctuating selection driven by global and local climatic conditions leads to stasis in breeding time in a migratory bird.

The origin of natural selection is linked to environmental heterogeneity, which influences variation in relative fitness among phenotypes. However, individuals in wild populations are exposed to a plethora of biotic and abiotic environmental factors. Surprisingly, the relative influence of multiple environmental conditions on the relative fitness of phenotypes has rarely been tested in wild populations. Identifying the main selection agent(s) is crucial when the target phenotype is tightly linked to reproduction and when temporal variation in selection is expected to affect evolutionary responses. By using individual-based data from a 29-year study of a short-lived migratory songbird, the pied flycatcher (Ficedula hypoleuca), we studied the relative influence of 28 temperature- and precipitation-based factors at local and global scales on selection on breeding time (egg laying) at the phenotypic level. Selection, estimated using the number of recruits as a proxy for fitness, penalized late breeders. Minimum temperatures in April and May were the environmental drivers that best explained selection on laying date. In particular, there was negative directional selection on laying date mediated by minimum temperature in April, being strongest in cold years. In addition, nonlinear selection on laying date was influenced by minimum temperatures in May, with selection on laying date changing from null to negative as the breeding season progressed. The intensity of selection on late breeders increased when minimum temperatures in May were highest. Our results illustrate the complex influence of environmental factors on selection on laying date in wild bird populations. Despite minimum temperature in April being the only variable that changed over time, its increase did not induce a shift in laying date in the population. In this songbird population, stabilizing selection has led to a three-decade stasis in breeding time. We suggest that variation in the effects of multiple climatic variables on selection may constrain phenotypic change.

Connecting the data landscape of long-term ecological studies: The SPI-Birds data hub.

The integration and synthesis of the data in different areas of science is drastically slowed and hindered by a lack of standards and networking programmes. Long-term studies of individually marked animals are not an exception. These studies are especially important as instrumental for understanding evolutionary and ecological processes in the wild. Furthermore, their number and global distribution provides a unique opportunity to assess the generality of patterns and to address broad-scale global issues (e.g. climate change). To solve data integration issues and enable a new scale of ecological and evolutionary research based on long-term studies of birds, we have created the SPI-Birds Network and Database (www.spibirds.org)-a large-scale initiative that connects data from, and researchers working on, studies of wild populations of individually recognizable (usually ringed) birds. Within year and a half since the establishment, SPI-Birds has recruited over 120 members, and currently hosts data on almost 1.5 million individual birds collected in 80 populations over 2,000 cumulative years, and counting. SPI-Birds acts as a data hub and a catalogue of studied populations. It prevents data loss, secures easy data finding, use and integration and thus facilitates collaboration and synthesis. We provide community-derived data and meta-data standards and improve data integrity guided by the principles of Findable, Accessible, Interoperable and Reusable (FAIR), and aligned with the existing metadata languages (e.g. ecological meta-data language). The encouraging community involvement stems from SPI-Bird's decentralized approach: research groups retain full control over data use and their way of data management, while SPI-Birds creates tailored pipelines to convert each unique data format into a standard format. We outline the lessons learned, so that other communities (e.g. those working on other taxa) can adapt our successful model. Creating community-specific hubs (such as ours, COMADRE for animal demography, etc.) will aid much-needed large-scale ecological data integration.

Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells.

Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO2, octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO2-mediated Cys-319 alkylation on ABL1 binding and found that OA-NO2 inhibits RAD51-ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51-ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile-based combinational therapies for TNBC.

Experimental evidence that matching habitat choice drives local adaptation in a wild population.

Matching habitat choice is a unique, flexible form of habitat choice based on self-assessment of local performance. This mechanism is thought to play an important role in adaptation and population persistence in variable environments. Nevertheless, the operation of matching habitat choice in natural populations remains to be unequivocally demonstrated. We investigated the association between body colour and substrate use by ground-perching grasshoppers (Sphingonotus azurescens) in an urban mosaic of dark and pale pavements, and then performed a colour manipulation experiment to test for matching habitat choice based on camouflage through background matching. Naturally, dark and pale grasshoppers occurred mostly on pavements that provided matching backgrounds. Colour-manipulated individuals recapitulated this pattern, such that black-painted and white-painted grasshoppers recaptured after the treatment aggregated together on the dark asphalt and pale pavement, respectively. Our study demonstrates that grasshoppers adjust their movement patterns to choose the substrate that confers an apparent improvement in camouflage given their individual-specific colour. More generally, our study provides unique experimental evidence of matching habitat choice as a driver of phenotype-environment correlations in natural populations and, furthermore, suggests that performance-based habitat choice might act as a mechanism of adaptation to changing environments, including human-modified (urban) landscapes.

Transcription with a laser: Radiation-damage-free diffraction of RNA Polymerase II crystals.

Well-diffracting crystals are essential to obtain relevant structural data that will lead to understanding of RNA Polymerase II (Pol II) transcriptional processes at a molecular level. Here we present a strategy to study Pol II crystals using negative stain transmission electron microscopy (TEM) and a methodology to optimize radiation damage free data collection using free electron laser (FEL) at the Linac Coherent Light Source (LCLS). The use of negative stain TEM allowed visualization and optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nano crystals bearing perfect lattices were seeded and used to grow larger crystals for FEL data collection. Moreover, the use of in house designed crystal loops together with ultra-violet (UV) microscopy for crystal detection facilitated data collection. Such strategy permitted collection of multiple crystals of radiation-free-damage data, resulting in the highest resolution of wild type (WT) Pol II crystals ever observed.

Variation in parasitoidism of Protocalliphora azurea (Diptera: Calliphoridae) by Nasonia vitripennis (Hymenoptera: Pteromalidae) in Spain.

Parasitoid wasps may act as hyperparasites and sometimes regulate the populations of their hosts by a top-down dynamic. Nasonia vitripennis (Walker, 1836) is a generalist gregarious parasitoid that parasitizes several host flies, including the blowfly Protocalliphora Hough, 1899 (Diptera, Calliphoridae), which in turn parasitizes bird nestlings. Nonetheless, the ecological factors underlying N. vitripennis prevalence and parasitoidism intensity on its hosts in natural populations are poorly understood. We have studied the prevalence of N. vitripennis in Protocalliphora azurea (Fallén, 1817) puparia parasitizing wild populations of pied flycatcher (Ficedula hypoleuca) and blue tit (Cyanistes caeruleus) birds in two Mediterranean areas in central and southern Spain. We found some evidence that the prevalence of N. vitripennis was higher in moist habitats in southern Spain. A host-dependent effect was found, since the greater the number of P. azurea puparia, the greater the probability and rate of parasitoidism by the wasp. Our results also suggest that N. vitripennis parasitizes more P. azurea puparia in blue tit nests than in pied flycatcher nests as a consequence of a higher load of these flies in the former. Based on the high prevalence of N. vitripennis in P. azurea puparia in nature, we propose that this wasp may regulate blowfly populations, with possible positive effects on the reproduction of both bird species.

Dysmorphic concern and behavioural impairment related to body image in adolescents.

This study examined a moderated mediation model testing whether dysmorphic concern is related to behaviour altered to change appearance. This relationship is potentially mediated by depressive symptomatology (dysphoria and self-esteem) and ideas of reference about "laughing, commenting" and "attention, appearance," and each mediated relationship moderated by sex. The sample was made up of 3377 adolescents from 12 to 18 years old (Mage  = 14.52; SD = 1.65, 56.5% girls). The results showed that dysphoria and ideas of reference about "laughing, commenting" and "attention, appearance" partially mediated the relationship between dysmorphic concern and behavioural impairment related to body image. The relationship with dysphoria was moderated by sex, such that the mediation effect was stronger in girls than in boys. This result implies that girls who are worried about some characteristic of their appearance and show dysphoria are at greater risk of altered behaviour involving avoidance or controlling their appearance than boys. In addition, a possible risk of body dysmorphic disorder (3.45% of the sample) was found, with very prominent hiding behaviour using clothing or control behaviours, such as frequent weighing and looking at oneself in the mirror too much. Implications of these findings are discussed.

A combined computational and experimental approach reveals the structure of a C/EBPß-Spi1 interaction required for IL1B gene transcription.

We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1ß, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPß) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPß leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPß DBDs, along with the C/EBPß C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPß-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPß's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPß to cognate DNA and in transcription of the C/EBPß-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.

Predicting protein targets for drug-like compounds using transcriptomics.

An expanded chemical space is essential for improved identification of small molecules for emerging therapeutic targets. However, the identification of targets for novel compounds is biased towards the synthesis of known scaffolds that bind familiar protein families, limiting the exploration of chemical space. To change this paradigm, we validated a new pipeline that identifies small molecule-protein interactions and works even for compounds lacking similarity to known drugs. Based on differential mRNA profiles in multiple cell types exposed to drugs and in which gene knockdowns (KD) were conducted, we showed that drugs induce gene regulatory networks that correlate with those produced after silencing protein-coding genes. Next, we applied supervised machine learning to exploit drug-KD signature correlations and enriched our predictions using an orthogonal structure-based screen. As a proof-of-principle for this regimen, top-10/top-100 target prediction accuracies of 26% and 41%, respectively, were achieved on a validation of set 152 FDA-approved drugs and 3104 potential targets. We then predicted targets for 1680 compounds and validated chemical interactors with four targets that have proven difficult to chemically modulate, including non-covalent inhibitors of HRAS and KRAS. Importantly, drug-target interactions manifest as gene expression correlations between drug treatment and both target gene KD and KD of genes that act up- or down-stream of the target, even for relatively weak binders. These correlations provide new insights on the cellular response of disrupting protein interactions and highlight the complex genetic phenotypes of drug treatment. With further refinement, our pipeline may accelerate the identification and development of novel chemical classes by screening compound-target interactions.

Optimal affinity ranking for automated virtual screening validated in prospective D3R grand challenges.

The goal of virtual screening is to generate a substantially reduced and enriched subset of compounds from a large virtual chemistry space. Critical in these efforts are methods to properly rank the binding affinity of compounds. Prospective evaluations of ranking strategies in the D3R grand challenges show that for targets with deep pockets the best correlations (Spearman ρ ~ 0.5) were obtained by our submissions that docked compounds to the holo-receptors with the most chemically similar ligand. On the other hand, for targets with open pockets using multiple receptor structures is not a good strategy. Instead, docking to a single optimal receptor led to the best correlations (Spearman ρ ~ 0.5), and overall performs better than any other method. Yet, choosing a suboptimal receptor for crossdocking can significantly undermine the affinity rankings. Our submissions that evaluated the free energy of congeneric compounds were also among the best in the community experiment. Error bars of around 1 kcal/mol are still too large to significantly improve the overall rankings. Collectively, our top of the line predictions show that automated virtual screening with rigid receptors perform better than flexible docking and other more complex methods.

Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.

Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90ß/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90ß]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.