Topical SCD-153, a 4-methyl itaconate prodrug, for the treatment of alopecia areata.

Alopecia areata is a chronic hair loss disorder that involves autoimmune disruption of hair follicles by CD8+  T cells. Most patients present with patchy hair loss on the scalp that improves spontaneously or with topical and intralesional steroids, topical minoxidil, or topical immunotherapy. However, recurrence of hair loss is common, and patients with extensive disease may require treatment with oral corticosteroids or oral Janus kinase (JAK) inhibitors, both of which may cause systemic toxicities with long-term use. Itaconate is an endogenous molecule synthesized in macrophages that exerts anti-inflammatory effects. To investigate the use of itaconate derivatives for treating alopecia areata, we designed a prodrug of 4-methyl itaconate (4-MI), termed SCD-153, with increased lipophilicity compared to 4-MI (CLogP 1.159 vs. 0.1442) to enhance skin and cell penetration. Topical SCD-153 formed 4-MI upon penetrating the stratum corneum in C57BL/6 mice and showed low systemic absorption. When added to human epidermal keratinocytes stimulated with polyinosinic-polycytidylic acid (poly I:C) or interferon (IFN)γ, SCD-153 significantly attenuated poly I:C-induced interleukin (IL)-6, Toll-like receptor 3, IL-1ß, and IFNß expression, as well as IFNγ-induced IL-6 expression. Topical application of SCD-153 to C57BL/6 mice in the resting (telogen) phase of the hair cycle induced significant hair growth that was statistically superior to vehicle (dimethyl sulfoxide), the less cell-permeable itaconate analogues 4-MI and dimethyl itaconate, and the JAK inhibitor tofacitinib. Our results suggest that SCD-153 is a promising topical candidate for treating alopecia areata.

Explicit versus implicit consideration of binding partners in protein-protein complex to elucidate intrinsic dynamics.

The binding of many proteins to their protein partners is tightly regulated via control of their relative intrinsic dynamics during the binding process, a phenomenon which can in turn be modulated. Therefore, investigating the intrinsic dynamics of proteins is necessary to understand function in a comprehensive way. By intrinsic dynamics herein, we principally refer to the vibrational signature of a protein molecule popularly obtained from normal modes or essential modes. For normal modes, one often considers that the molecule under investigation is a collection of springs in a solvent-free or implicit-solvent medium. In the context of a protein-binding partner, the analysis of vibration of the target protein is often complicated due to molecular interaction within the complex. Generally, it is assumed that the isolated bound conformation of the target protein captures the implicit effect of the binding partner on the intrinsic dynamics, therefore suggesting that any influence of the partner molecule is also already integrated. Such an assumption allows large-scale studies of the conservation of protein flexibility. However, in cases where a partner protein directly influences the vibration of the target via critical contacts at the protein-protein interface, the above assumption falls short of providing a detailed view. In this review article, we discuss the implications of considering the dynamics of a protein in a protein-protein complex, as modelled implicitly and explicitly with methods dependent on elastic network models. We further propose how such an explicit consideration can be applied to understand critical protein-protein contacts that can be targeted in future studies.

Protocol for Retrieving Three-Dimensional Biological Shapes for a Few XFEL Single-Particle Diffraction Patterns.

X-ray free-electron laser (XFEL) scattering promises to probe single biomolecular complexes without crystallization, enabling the study of biomolecular structures under near-physiological conditions at room temperature. However, such structural determination of biomolecules is extremely challenging thus far. In addition to the large numbers of diffraction patterns required, the orientation of each diffraction pattern needs to be accurately estimated and the missing phase information needs to be recovered for three-dimensional (3D) structure reconstruction. Given the current limitations to the amount and resolution of the data available from single-particle XFEL scattering experiments, we propose an alternative approach to find plausible 3D biological shapes from a limited number of diffraction patterns to serve as a starting point for further analyses. In our proposed strategy, small sets of input (e.g., five) XFEL diffraction patterns were matched against a library of diffraction patterns simulated from 1628 electron microscopy (EM) models to find potential matching 3D models that are consistent with the input diffraction patterns. This approach was tested for three example cases: EMD-3457 (Thermoplasma acidophilum 20S proteasome), EMD-5141 (Escherichia coli 70S ribosome complex), and EMD-5152 (budding yeast Nup84 complex). We observed that choosing the best strategy to define matching regions on the diffraction patterns is critical for identifying correctly matching diffraction patterns. While increasing the number of input diffraction patterns improved the matches in some cases, we found that the resulting matches are more dependent on the uniqueness or complexity of the shape as captured in the individual input diffraction patterns and the availability of a similar 3D biological shape in the search library. The protocol could be useful for finding candidate models for a limited amount of low-resolution data, even when insufficient for reconstruction, performing a quick exploration of new data upon collection, and the analysis of the conformational heterogeneity of the particle of interest as captured within the diffraction patterns.

Integrative/Hybrid Modeling Approaches for Studying Biomolecules.

The structural and dynamical characterization of biomolecules holds central importance in the endeavor to understand the molecular mechanisms regulating living systems. However, owing to the inherent heterogeneity of biomolecular interactions within cells, it is often difficult to understand the overall structure and dynamics of biomolecules using any experimental method in isolation. In this regard, hybrid methods that combine data from multiple experiments to generate a comprehensive model of biomolecular complexes have gained prominence in the last few years. In this article, we discuss the advancements in hybrid methods, with a particular focus on the role of computation in their development and application. We further outline the future directions that hybrid methods are likely to take, regarding the advancements in techniques such as X-ray free-electron laser single- particle imaging, and electron cryo-tomography. Finally, we conclude the review by highlighting the future goals of broader consensus and collaboration within the integrative/hybrid structural biology community and for disseminating the data generated by hybrid modeling efforts.

Computational Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy of Single-particle Cryo-electron Microscopy Maps.

Cryo-electron microscopy (cryo-EM) single-particle analysis has come a long way in achieving atomic-level resolution when imaging biomolecules. To obtain the best possible three-dimensional (3D) structure in cryo-EM, many parameters have to be carefully considered. Here we address the often-overlooked parameter of the pixel size, which describes the magnification of the image produced by the experiment. While efforts are made to refine and validate this parameter in the analysis of cryo-EM experimental data, there is no systematic protocol in place. Since the pixel size parameter can have an impact on the resolution and accuracy of a cryo-EM map, and the atomic resolution 3D structure models derived from it, we propose a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM maps at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP (generalized orientation-dependent, all-atom statistical potential) score, which we found to perform better than other commonly used functions, such as Molprobity and the correlation coefficient from refinement. Finally, we describe the efficacy of this protocol in retrieving appropriate pixel sizes for several examples; simulated data based on yeast elongation factor 2 and experimental data from Gro-EL chaperone, beta-galactosidase, and the TRPV1 ion channel.

Searching for 3D structural models from a library of biological shapes using a few 2D experimental images.

BACKGROUND: Advancements in biophysical experimental techniques have pushed the limits in terms of the types of phenomena that can be characterized, the amount of data that can be produced and the resolution at which we can visualize them. Single particle techniques such as Electron Microscopy (EM) and X-ray free electron laser (XFEL) scattering require a large number of 2D images collected to resolve three-dimensional (3D) structures. In this study, we propose a quick strategy to retrieve potential 3D shapes, as low-resolution models, from a few 2D experimental images by searching a library of 2D projection images generated from existing 3D structures. RESULTS: We developed the protocol to assemble a non-redundant set of 3D shapes for generating the 2D image library, and to retrieve potential match 3D shapes for query images, using EM data as a test. In our strategy, we disregard differences in volume size, giving previously unknown structures and conformations a greater number of 3D biological shapes as possible matches. We tested the strategy using images from three EM models as query images for searches against a library of 22750 2D projection images generated from 250 random EM models. We found that our ability to identify 3D shapes that match the query images depends on how complex the outline of the 2D shapes are and whether they are represented in the search image library. CONCLUSIONS: Through our computational method, we are able to quickly retrieve a 3D shape from a few 2D projection images. Our approach has the potential for exploring other types of 2D single particle structural data such as from XFEL scattering experiments, for providing a tool to interpret low-resolution data that may be insufficient for 3D reconstruction, and for estimating the mixing of states or conformations that could exist in such experimental data.

Conservation of intrinsic dynamics in proteins-what have computational models taught us?

The intrinsic dynamics of proteins has been suggested to be the most conserved compared to its sequence or structure. As such, the contributing factors to the conservation of dynamics have yet to be determined definitively. Some have suggested that function drives the conservation of protein flexibility, while others have indicated that the overall topology determines protein flexibility patterns. In general, many characteristic features of protein flexibility can be derived from simple coarse-grained models whose success rests on the link between protein local packing density and flexibility. Those models have revealed the evolutionary conservation of protein flexibility and have given us insights into the slow dynamics required for protein function and mechanistic insight into the allosteric effect.

Similarity in Shape Dictates Signature Intrinsic Dynamics Despite No Functional Conservation in TIM Barrel Enzymes.

The conservation of the intrinsic dynamics of proteins emerges as we attempt to understand the relationship between sequence, structure and functional conservation. We characterise the conservation of such dynamics in a case where the structure is conserved but function differs greatly. The triosephosphate isomerase barrel fold (TBF), renowned for its 8 ß-strand-α-helix repeats that close to form a barrel, is one of the most diverse and abundant folds found in known protein structures. Proteins with this fold have diverse enzymatic functions spanning five of six Enzyme Commission classes, and we have picked five different superfamily candidates for our analysis using elastic network models. We find that the overall shape is a large determinant in the similarity of the intrinsic dynamics, regardless of function. In particular, the ß-barrel core is highly rigid, while the α-helices that flank the ß-strands have greater relative mobility, allowing for the many possibilities for placement of catalytic residues. We find that these elements correlate with each other via the loops that link them, as opposed to being directly correlated. We are also able to analyse the types of motions encoded by the normal mode vectors of the α-helices. We suggest that the global conservation of the intrinsic dynamics in the TBF contributes greatly to its success as an enzymatic scaffold both through evolution and enzyme design.

Comparing the intrinsic dynamics of multiple protein structures using elastic network models.

BACKGROUND: Elastic network models (ENMs) are based on the simple idea that a protein can be described as a set of particles connected by springs, which can then be used to describe its intrinsic flexibility using, for example, normal mode analysis. Since the introduction of the first ENM by Monique Tirion in 1996, several variants using coarser protein models have been proposed and their reliability for the description of protein intrinsic dynamics has been widely demonstrated. Lately an increasing number of studies have focused on the meaning of slow dynamics for protein function and its potential conservation through evolution. This leads naturally to comparisons of the intrinsic dynamics of multiple protein structures with varying levels of similarity. SCOPE OF REVIEW: We describe computational strategies for calculating and comparing intrinsic dynamics of multiple proteins using elastic network models, as well as a selection of examples from the recent literature. MAJOR CONCLUSIONS: The increasing interest for comparing dynamics across protein structures with various levels of similarity, has led to the establishment and validation of reliable computational strategies using ENMs. Comparing dynamics has been shown to be a viable way for gaining greater understanding for the mechanisms employed by proteins for their function. Choices of ENM parameters, structure alignment or similarity measures will likely influence the interpretation of the comparative analysis of protein motion. GENERAL SIGNIFICANCE: Understanding the relation between protein function and dynamics is relevant to the fundamental understanding of protein structure-dynamics-function relationship. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

WEBnm@ v2.0: Web server and services for comparing protein flexibility.

BACKGROUND: Normal mode analysis (NMA) using elastic network models is a reliable and cost-effective computational method to characterise protein flexibility and by extension, their dynamics. Further insight into the dynamics-function relationship can be gained by comparing protein motions between protein homologs and functional classifications. This can be achieved by comparing normal modes obtained from sets of evolutionary related proteins. RESULTS: We have developed an automated tool for comparative NMA of a set of pre-aligned protein structures. The user can submit a sequence alignment in the FASTA format and the corresponding coordinate files in the Protein Data Bank (PDB) format. The computed normalised squared atomic fluctuations and atomic deformation energies of the submitted structures can be easily compared on graphs provided by the web user interface. The web server provides pairwise comparison of the dynamics of all proteins included in the submitted set using two measures: the Root Mean Squared Inner Product and the Bhattacharyya Coefficient. The Comparative Analysis has been implemented on our web server for NMA, WEBnm@, which also provides recently upgraded functionality for NMA of single protein structures. This includes new visualisations of protein motion, visualisation of inter-residue correlations and the analysis of conformational change using the overlap analysis. In addition, programmatic access to WEBnm@ is now available through a SOAP-based web service. Webnm@ is available at http://apps.cbu.uib.no/webnma . CONCLUSION: WEBnm@ v2.0 is an online tool offering unique capability for comparative NMA on multiple protein structures. Along with a convenient web interface, powerful computing resources, and several methods for mode analyses, WEBnm@ facilitates the assessment of protein flexibility within protein families and superfamilies. These analyses can give a good view of how the structures move and how the flexibility is conserved over the different structures.

Evolution of oligomeric state through allosteric pathways that mimic ligand binding.

Evolution and design of protein complexes are almost always viewed through the lens of amino acid mutations at protein interfaces. We showed previously that residues not involved in the physical interaction between proteins make important contributions to oligomerization by acting indirectly or allosterically. In this work, we sought to investigate the mechanism by which allosteric mutations act, using the example of the PyrR family of pyrimidine operon attenuators. In this family, a perfectly sequence-conserved helix that forms a tetrameric interface is exposed as solvent-accessible surface in dimeric orthologs. This means that mutations must be acting from a distance to destabilize the interface. We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. Finally, we show that the key mutations introduce conformational changes equivalent to the conformational shift between the free versus nucleotide-bound conformations of the proteins.

Hippophae leaf extract concentration regulates antioxidant and prooxidant effects on DNA.

Extracts from Hippophae leaves constitute some commonly consumed beverages such as tea and wine. We had developed an extract of Hippophae leaves (SBL-1), which was rich in quercetin, had antimutagenic effects, radioprotective effects, and countered radiation-induced gene conversion in Saccharomyces cerevisiae. This study was designed to investigate the action of SBL-1 on guanine cytosine (GC)-rich nascent and mouse genomic DNA in vitro. The human and mouse liver DNA have about 43% GC content. Our results showed that at small concentration SBL-1 protected nascent as well as genomic DNA, while at large concentration SBL-1 damaged both types of DNA. The concentration of SBL-1 that protected DNA also demonstrated higher free radical scavenging activity. The reducing power of SBL-1 was greater than its free radical scavenging activity. The greater reducing power may have reduced the trace metals present in the SBL-1, leading to generation of hydroxyl radicals via Fenton reaction. The increased proportion of unscavenged hydroxyl radicals with increase in SBL-1 concentration may have been responsible for DNA damage or prooxidant effect of SBL-1 in vitro. This study suggests that the dietary supplements prepared from Hippophae should have low metal content.

Diclofenac-induced biochemical and histopathological changes in white leghorn birds (Gallus domesticus).

OBJECTIVE: To evaluate diclofenac-induced biochemical and histopathological changes in White Leghorn birds. MATERIALS AND METHODS: Six-week-old birds were equally divided into three groups of six birds each. Group I served as control and received vehicle orally. The birds of Group II and III were orally administered with a single low (2 mg/kg) and high dose (20 mg/kg) of diclofenac sodium, respectively, and were observed for 7 days. The acute toxicity was assessed by observing the clinical signs and symptoms, mortality, alterations in blood biochemistry, and necropsy findings. RESULTS: The birds of Group II showed only mild symptoms of diarrhea. In Group III, 50% of birds died in between 24 and 36 h post-treatment showing the symptoms of segregatory behavior, lethargy, terminal anorexia, and severe bloody diarrhea. The birds of Group II and the surviving birds of Group III showed a significantly (P<0.05) increased plasma uric acid, creatinine and plasma glutamic pyruvic transaminase (PGPT), and decreased total protein and albumin at 12 and 24 h post-treatment which returned to the normal levels at 36 h post-treatment. The dead birds of the high-dose group also showed similar pattern of biochemical changes at 12 and 24 h post-treatment and revealed extensive visceral gout with characteristic histopathological lesions in liver, kidney, heart, spleen, and intestine on post-mortem. CONCLUSION: The results indicate that diclofenac sodium has hepatotoxic, nephrotoxic, and visceral gout inducing potentials in White Leghorn birds, especially at higher dose.