Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4.

IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments.

SBMOpenMM: A Builder of Structure-Based Models for OpenMM.

Molecular dynamics (MD) simulations have become a standard tool to correlate the structure and function of biomolecules and significant advances have been made in the study of proteins and their complexes. A major drawback of conventional MD simulations is the difficulty and cost of obtaining converged results, especially when exploring potential energy surfaces containing considerable energy barriers. This limits the wide use of MD calculations to determine the thermodynamic properties of biomolecular processes. Indeed, this is true when considering the conformational entropy of such processes, which is ultimately critical in assessing the simulations' convergence. Alternatively, a wide range of structure-based models (SBMs) has been used in the literature to unravel the basic mechanisms of biomolecular dynamics. These models introduce simplifications that focus on the relevant aspects of the physical process under study. Because of this, SBMs incorporate the need to modify the force field definition and parameters to target specific biophysical simulations. Here we introduce SBMOpenMM, a Python library to build force fields for SBMs, that uses the OpenMM framework to create and run SBM simulations. The code is flexible and user-friendly and profits from the high customizability and performance provided by the OpenMM platform.

Computational exploration of the binding mode of heme-dependent stimulators into the active catalytic domain of soluble guanylate cyclase.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an "in silico" approach. Homology models of the catalytic domain of sGC in "inactive" or "active" conformations were constructed using the structure of previously described crystals of the catalytic domains of "inactive" sGCs (2WZ1, 3ET6) and of "active" adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 µs. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of "inactive" and "active" conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the "active" structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop ß2 -ß3 , does not overlap with the forskolin binding site on adenylate cyclases. Proteins 2016; 84:1534-1548. © 2016 Wiley Periodicals, Inc.

Ancient Loss of Catalytic Selenocysteine Spurred Convergent Adaptation in a Mammalian Oxidoreductase.

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.

Identification of Aggregation Mechanism of Acetylated PHF6* and PHF6 Tau Peptides Based on Molecular Dynamics Simulations and Markov State Modeling.

The microtubule-associated protein tau (MAPT) has a critical role in the development and preservation of the nervous system. However, tau's dysfunction and accumulation in the human brain can lead to several neurodegenerative diseases, such as Alzheimer's disease, Down's syndrome, and frontotemporal dementia. The microtubule binding (MTB) domain plays a significant, important role in determining the tau's pathophysiology, as the core of paired helical filaments PHF6* (275VQIINK280) and PHF6 (306VQIVYK311) of R2 and R3 repeat units, respectively, are formed in this region, which promotes tau aggregation. Post-translational modifications, and in particular lysine acetylation at K280 of PHF6* and K311 of PHF6, have been previously established to promote tau misfolding and aggregation. However, the exact aggregation mechanism is not known. In this study, we established an atomic-level nucleation-extension mechanism of the separated aggregation of acetylated PHF6* and PHF6 hexapeptides, respectively, of tau. We show that the acetylation of the lysine residues promotes the formation of ß-sheet enriched high-ordered oligomers. The Markov state model analysis of ac-PHF6* and ac-PHF6 aggregation revealed the formation of an antiparallel dimer nucleus which could be extended from both sides in a parallel manner to form mixed-oriented and high-ordered oligomers. Our study describes the detailed mechanism for acetylation-driven tau aggregation, which provides valuable insights into the effect of post-translation modification in altering the pathophysiology of tau hexapeptides.

CDK6 is activated by the atypical cyclin I to promote E2F-mediated gene expression and cancer cell proliferation.

Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.

New model of cystic fibrosis transmembrane conductance regulator proposes active channel-like conformation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter, functioning as a chloride channel critical for fluid homeostasis in multiple organs. Disruption of CFTR function is associated with cystic fibrosis making it an attractive therapeutic target. In addition, CFTR blockers are being developed as potential antidiarrheals. CFTR drug discovery is hampered by the lack of high resolution structural data, and considerable efforts have been invested in modeling the channel structure. Although previously published CFTR models that have been made publicly available mostly agree with experimental data relating to the overall structure, they present the channel in an outward-facing conformation that does not agree with expected properties of a "channel-like" structure. Here, we make available a model of CFTR in such a "channel-like" conformation, derived by a unique modeling approach combining restrained homology modeling and ROSETTA refinement. In contrast to others, the present model is in agreement with expected channel properties such as pore shape, dimensions, solvent accessibility, and experimentally derived distances. We have used the model to explore the interaction of open channel blockers within the pore, revealing a common binding mode and ionic interaction with K95, in agreement with experimental data. The binding-site was further validated using a virtual screening enrichment experiment, suggesting the model might be suitable for drug discovery. In addition, we subjected the model to a molecular dynamics simulation, revealing previously unaddressed salt-bridge interactions that may be important for structure stability and pore-lining residues that may take part in Cl(-) conductance.

New Mutations in HFE2 and TFR2 Genes Causing Non HFE-Related Hereditary Hemochromatosis.

Hereditary hemochromatosis (HH) is an iron metabolism disease clinically characterized by excessive iron deposition in parenchymal organs such as liver, heart, pancreas, and joints. It is caused by mutations in at least five different genes. HFE hemochromatosis is the most common type of hemochromatosis, while non-HFE related hemochromatosis are rare cases. Here, we describe six new patients of non-HFE related HH from five different families. Two families (Family 1 and 2) have novel nonsense mutations in the HFE2 gene have novel nonsense mutations (p.Arg63Ter and Asp36ThrfsTer96). Three families have mutations in the TFR2 gene, one case has one previously unreported mutation (Family A-p.Asp680Tyr) and two cases have known pathogenic mutations (Family B and D-p.Trp781Ter and p.Gln672Ter respectively). Clinical, biochemical, and genetic data are discussed in all these cases. These rare cases of non-HFE related hereditary hemochromatosis highlight the importance of an earlier molecular diagnosis in a specialized center to prevent serious clinical complications.

Computational study on the carboligation reaction of acetohidroxyacid synthase: new approach on the role of the HEThDP- intermediate.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl-thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP(-)). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP(-) has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP(-)) and a second pyruvate molecule. Our results suggest that additional acid-base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid-base properties of the HEThDP(-) intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP(-) intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation.

Alzheimer's disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Analysis of electrostatic contributions to the selectivity of interactions between RING-finger domains and ubiquitin-conjugating enzymes.

The zinc-coordinated protein motifs known as RING-finger domains, present on a class of ubiquitin ligases (E3's), recruit ubiquitin-conjugating enzymes (E2s), tethering them to substrate proteins for covalent modification with ubiquitin. Each RING-finger domain can recruit different E2s, and these interactions are frequently selective, in that certain RING-finger domains associate preferentially with certain E2s. This selectivity acquires particular biological relevance when the recruited E2s exert specialized functions. We have explored the determinants that specify the presence or absence of experimentally detectable interaction between two RING-finger domains, those on RNF11 and RNF103, and two E2s, UBC13, a specialized E2 that catalyzes ubiquitin chain elongation through Lys63 of ubiquitin, and UbcH7, which mediates polyubiquitylation through Lys48. Through the iterative use of computational predictive tools and experimental validations, we have found that these interactions and their selectivity are partly governed by the combinations of electrostatic interactions linking specific residues of the contact interfaces. Our analysis also predicts that the main determinants of selectivity of these interactions reside on the RING-finger domains, rather than on the E2s. The application of some of these rules of interaction selectivity has permitted us to experimentally manipulate the selectivity of interaction of the RING-finger domain-E2 pairs under study.

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

The role of residue stability in transient protein-protein interactions involved in enzymatic phosphate hydrolysis. A computational study.

Finding why protein-protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so-called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S-LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate "hot spots" for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper "energetic" orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above-mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient PPIs.

Antiretroviral therapy suppressed participants with low CD4+ T-cell counts segregate according to opposite immunological phenotypes.

BACKGROUND: The failure to increase CD4 T-cell counts in some antiretroviral therapy suppressed participants (immunodiscordance) has been related to perturbed CD4 T-cell homeostasis and impacts clinical evolution. METHODS: We evaluated different definitions of immunodiscordance based on CD4 T-cell counts (cutoff) or CD4 T-cell increases from nadir value (ΔCD4) using supervised random forest classification of 74 immunological and clinical variables from 196 antiretroviral therapy suppressed individuals. Unsupervised clustering was performed using relevant variables identified in the supervised approach from 191 individuals. RESULTS: Cutoff definition of CD4 cell count 400 cells/µl performed better than any other definition in segregating immunoconcordant and immunodiscordant individuals (85% accuracy), using markers of activation, nadir and death of CD4 T cells. Unsupervised clustering of relevant variables using this definition revealed large heterogeneity between immunodiscordant individuals and segregated participants into three distinct subgroups with distinct production, programmed cell-death protein-1 (PD-1) expression, activation and death of T cells. Surprisingly, a nonnegligible number of immunodiscordant participants (22%) showed high frequency of recent thymic emigrants and low CD4 T-cell activation and death, very similar to immunoconcordant participants. Notably, human leukocyte antigen - antigen D related (HLA-DR) PD-1 and CD45RA expression in CD4 T cells allowed reproducing subgroup segregation (81.4% accuracy). Despite sharp immunological differences, similar and persistently low CD4 values were maintained in these participants over time. CONCLUSION: A cutoff value of CD4 T-cell count 400 cells/µl classified better immunodiscordant and immunoconcordant individuals than any ΔCD4 classification. Immunodiscordance may present several, even opposite, immunological patterns that are identified by a simple immunological follow-up. Subgroup classification may help clinicians to delineate diverse approaches that may be needed to boost CD4 T-cell recovery.

Towards a MIP-based alignment and docking in computer-aided drug design.

Structural alignment of ligands in their biological conformation is a crucial step in the building of pharmacophoric models in structure-based drug design. In addition, docking algorithms are limited in some cases by the quality of the scoring functions and the limited flexibility of the environment that the different programs allow. On the other hand, GRID molecular interaction potentials (MIPs) have been used for a long time in 3D-QSAR studies. However, in most of these studies the alignment of the molecules is performed on the basis of geometrical or physico-chemical criteria that differ from the MIPs used in the partial least squares statistical analysis. We have previously developed a method to use the same scoring function for the molecular alignment and for 3D-QSAR studies. This methodology, based on the use of GRID potentials, consists in the weighted averaging of similarities of the relevant MIPs of the molecules to be aligned. Here we present a method to obtain the weights for the different GRID probes in the average based on the structural information on protein-ligand complexes for relevant systems. The method, implemented in MIPSIM, is shown to yield good accuracy in the prediction of the alignments for two systems: a set of three inhibitors of dihydrofolate reductase and a set of fifteen non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs). The smooth GRID potentials are shown to capture the flexible character of the active site, as opposed to traditional docking scoring energy functions.

TRIFORCE: Tessellated Semianalytical Solvent Exposed Surface Areas and Derivatives.

We present a new approach to the calculation of solvent-accessible surface areas of molecules with potential application to surface area based methods for determination of solvation free energies. As in traditional analytical and statistical approaches, this new algorithm, called TRIFORCE, reports both component areas and derivatives as a function of the atomic coordinates and radii. Unique to TRIFORCE are the rapid and scalable approaches for the determination of sphere intersection points and numerical estimation of the surface areas, derivatives, and other properties that can be associated with the surface area facets. The algorithm performs a special tessellation and semianalytical integration that uses a precomputed look-up table. This provides a simple way to balance numerical accuracy and memory usage. TRIFORCE calculates derivatives in the same manner, enabling application in force-dependent activities such as molecular geometry minimization. TRIFORCE is available free of charge for academic purposes as both a C++ library, which can be directly interfaced to existing molecular simulation packages, and a web-accessible application.

Methylglyoxal produced by amyloid-ß peptide-induced nitrotyrosination of triosephosphate isomerase triggers neuronal death in Alzheimer's disease.

Amyloid-ß peptide (Aß) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aß42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aß42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aß action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aß oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.

Chromatin-bound IκBα regulates a subset of polycomb target genes in differentiation and cancer.

IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation.

A truncated form of IKKα is responsible for specific nuclear IKK activity in colorectal cancer.

Nuclear IKKα regulates gene transcription by phosphorylating specific substrates and has been linked to cancer progression and metastasis. However, the mechanistic connection between tumorigenesis and IKKα activity remains poorly understood. We have now analyzed 288 human colorectal cancer samples and found a significant association between the presence of nuclear IKK and malignancy. Importantly, the nucleus of tumor cells contains an active IKKα isoform with a predicted molecular weight of 45 kDa (p45-IKKα) that includes the kinase domain but lacks several regulatory regions. Active nuclear p45-IKKα forms a complex with nonactive IKKα and NEMO that mediates phosphorylation of SMRT and histone H3. Proteolytic cleavage of FL-IKKα into p45-IKKα is required for preventing the apoptosis of CRC cells in vitro and sustaining tumor growth in vivo. Our findings identify a potentially druggable target for treating patients with advance refractory CRC.

Multiscale molecular dynamics of protein aggregation.

The 60's gave birth to the practical implementation of classical mechanics to unravel the dynamics and energetics of biomolecules. In the 70's the use of generalized force fields and more advanced integrative solutions to the microscopic understanding of nature (like hybrid QM/MM) were introduced. During the 80's, algorithms to obtain free energy values were further developed and in the 90's practical integration schemes of molecular mechanics force fields with other levels of detail (QM on one extreme and advances in implicit solvation on the other) were implemented in widely spread software. In the first decade of the XXIst century a considerable effort has been put in two seemingly discordant models for the simulation of biomolecules. On the one hand, extraordinary advances in computing technologies (both in terms of processor power and of new efficient parallel and distributed computing schemas) have allowed researchers to deal with bigger systems and longer simulations, reaching molecular processes including millions of particles or lying in the microsecond scale. On the other hand, the realization that the relevant answers to many biomolecular problems are not homogeneously distributed through the molecular structure, something already envisioned by the QM/MM pioneers more than three decades ago, has led researchers to find smart ways of putting different emphases on different ranges of the spatial or system time scale. In this context, e.g., molecular aggregation represents a paradigm for multiscalability, as molecular recognition can be understood with simple (semi-)macroscopic terms when the two fragments are far apart, while the atomic interactions need to be considered in full detail upon close distances. In this manuscript the current status of the techniques that use multiple scale representations of biomolecules are reviewed, and the findings are synthesized in a modular schema that can be extensively used when studying aggregation processes. It is shown that a smart alternative to brute force and massive computation of uninteresting regions in the all atom potential energy surface is the consideration of a simplified reference potential, explored thoroughly in the relevant regions, combined with a free energy perturbation approach that transforms this simple representation to a full atom representation.