A highly conserved program of neuronal microexons is misregulated in autistic brains.

Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.

A system-view of Bordetella pertussis booster vaccine responses in adults primed with whole-cell versus acellular vaccine in infancy.

The increased incidence of whooping cough worldwide suggests that current vaccination against Bordetella pertussis infection has limitations in quality and duration of protection. The resurgence of infection has been linked to the introduction of acellular vaccines (aP), which have an improved safety profile compared with the previously used whole-cell (wP) vaccines. To determine immunological differences between aP and wP priming in infancy, we performed a systems approach of the immune response to booster vaccination. Transcriptomic, proteomic, cytometric, and serologic profiling revealed multiple shared immune responses with different kinetics across cohorts, including an increase of blood monocyte frequencies and strong antigen-specific IgG responses. Additionally, we found a prominent subset of aP-primed individuals (30%) with a strong differential signature, including higher levels of expression for CCL3, NFKBIA, and ICAM1. Contrary to the wP individuals, this subset displayed increased PT-specific IgE responses after boost and higher antigen-specific IgG4 and IgG3 antibodies against FHA and FIM2/3 at baseline and after boost. Overall, the results show that, while broad immune response patterns to Tdap boost overlap between aP- and wP-primed individuals, a subset of aP-primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.

Multi-constraint computational design suggests that native sequences of germline antibody H3 loops are nearly optimal for conformational flexibility.

The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen-binding site can significantly enlarge the repertoire. Among the six complementarity determining regions (CDRs) that generally comprise the binding site, the CDR H3 loop is particularly variable. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi-constraint computational design strategy, along with the Rosetta scoring function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen-bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by preorganizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity.

Host Transcriptomics as a Tool to Identify Diagnostic and Mechanistic Immune Signatures of Tuberculosis.

Tuberculosis (TB) is a major infectious disease worldwide, and is associated with several challenges for control and eradication. First, more accurate diagnostic tools that better represent the spectrum of infection states are required; in particular, identify the latent TB infected individuals with high risk of developing active TB. Second, we need to better understand, from a mechanistic point of view, why the immune system is unsuccessful in some cases for control and elimination of the pathogen. Host transcriptomics is a powerful approach to identify both diagnostic and mechanistic immune signatures of diseases. We have recently reported that optimal study design for these two purposes should be guided by different sets of criteria. Here, based on already published transcriptomics signatures of tuberculosis, we further develop these guidelines and identify additional factors to consider for obtaining diagnostic vs. mechanistic signatures in terms of cohorts, samples, data generation and analysis. Diagnostic studies should aim to identify small disease signatures with high discriminatory power across all affected populations, and against similar pathologies to TB. Specific focus should be made on improving the diagnosis of infected individuals at risk of developing active disease. Conversely, mechanistic studies should focus on tissues biopsies, immune relevant cell subsets, state of the art transcriptomic techniques and bioinformatics tools to understand the biological meaning of identified gene signatures that could facilitate therapeutic interventions. Finally, investigators should ensure their data are made publicly available along with complete annotations to facilitate metadata and cross-study analyses.

Dengue-specific CD8+ T cell subsets display specialized transcriptomic and TCR profiles.

Accumulating evidence demonstrates that CD8+ T cells contribute to protection from severe dengue virus (DENV) disease and vaccine efficacy. Nevertheless, molecular programs associated with DENV-specific CD8+ T cell subsets have not been defined. Here, we studied the transcriptomic profiles of human DENV-specific CD8+ T cells isolated after stimulation with DENV epitopes from donors who had been infected with DENV multiple times and would therefore be expected to have significant levels of adaptive immunity. We found that DENV-specific CD8+ T cells mainly consisted of effector memory subsets, namely CD45RA-CCR7- effector memory (Tem) and CD45RA+CCR7- effector memory re-expressing CD45RA (Temra) cells, which enacted specific gene expression profiles upon stimulation with cognate antigens. DENV-specific CD8+ T cell subsets in general, and Temra cells in particular, were fully activated and polyfunctional, yet associated with relatively narrow transcriptional responses. Furthermore, we found that DENV-specific CD8+ Tem and Temra cells showed some unique T cell receptor features in terms of overlap and variable (V) gene usage. This study provides a transcriptomic definition of DENV-specific activated human CD8+ T cell subsets and defines a benchmark profile that vaccine-specific responses could aim to reproduce.

Circulating T cell-monocyte complexes are markers of immune perturbations.

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

Prediction of transition metal-binding sites from apo protein structures.

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.

Th1/Th17 polarization persists following whole-cell pertussis vaccination despite repeated acellular boosters.

In the mid-1990s, whole-cell pertussis (wP) vaccines were associated with local and systemic adverse events that prompted their replacement with acellular pertussis (aP) vaccines in many high-income countries. In the past decade, rates of pertussis disease have increased in children receiving only aP vaccines. We compared the immune responses to aP boosters in individuals who received their initial doses with either wP or aP vaccines using activation-induced marker (AIM) assays. Specifically, we examined pertussis-specific memory CD4+ T cell responses ex vivo, highlighting a type 2/Th2 versus type 1/Th1 and Th17 differential polarization as a function of childhood vaccination. Remarkably, after a contemporary aP booster, cells from donors originally primed with aP were (a) associated with increased IL-4, IL-5, IL-13, IL-9, and TGF-ß and decreased IFN-γ and IL-17 production, (b) defective in their ex vivo capacity to expand memory cells, and (c) less capable of proliferating in vitro. These differences appeared to be T cell specific, since equivalent increases of antibody titers and plasmablasts after aP boost were seen in both groups. In conclusion, our data suggest that there are long-lasting effects and differences in polarization and proliferation of T cell responses in adults originally vaccinated with aP compared with those that initially received wP, despite repeated acellular boosters.

SPACE: a suite of tools for protein structure prediction and analysis based on complementarity and environment.

We describe a suite of SPACE tools for analysis and prediction of structures of biomolecules and their complexes. LPC/CSU software provides a common definition of inter-atomic contacts and complementarity of contacting surfaces to analyze protein structure and complexes. In the current version of LPC/CSU, analyses of water molecules and nucleic acids have been added, together with improved and expanded visualization options using Chime or Java based Jmol. The SPACE suite includes servers and programs for: structural analysis of point mutations (MutaProt); side chain modeling based on surface complementarity (SCCOMP); building a crystal environment and analysis of crystal contacts (CryCo); construction and analysis of protein contact maps (CMA) and molecular docking software (LIGIN). The SPACE suite is accessed at http://ligin.weizmann.ac.il/space.

Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA.

The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56+ TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56+ TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens.

Flexibility of metal binding sites in proteins on a database scale.

Protein metal binding sites in the pre-bound (apo) state, and their rearrangements upon metal binding were not analyzed previously at a database scale. Such a study may provide valuable information for metal binding site prediction and design. A high resolution, nonredundant dataset of 210 metal binding sites was created, containing all available representatives of apo-holo pairs for the most populated metals in the PDB. More than 40% of the sites underwent rearrangements upon metal binding. In 30 cases rearrangements involved the backbone. The tendency for side-chain rearrangement inversely correlates with the number of first-shell residues. Analysis of side-chain reorientations as a result of metal binding showed that in 95% of the rigid-backbone binding sites at most one side chain moved. Thus, in general, part of the first coordination shell is already in place in the pre-bound form. The frequencies of side-chain reorientation directly correlated with metal ligand flexibility and solvent accessibility in the apo state.

Conserved positions for ribose recognition: importance of water bridging interactions among ATP, ADP and FAD-protein complexes.

Analysis of the spatial arrangement of protein and water atoms that form polar interactions with ribose has been performed for a structurally non-redundant dataset of ATP, ADP and FAD-protein complexes. The 26 ligand-protein structures were separated into two groups corresponding to the most populated furanose ring conformations (N and S-domains). Four conserved positions were found for S-domain protein-ligand complexes and five for N-domain complexes. Multiple protein folds and secondary structural elements were represented at a single conserved position. The following novel points were revealed: (i) Two complementary positions sometimes combine to describe a putative atomic spatial location for a specific conserved binding spot. (ii) More than one third of the interactions scored were water-mediated. Thus, conserved spatial positions rich in water atoms are a significant feature of ribose-protein complexes.

Assessment of flexible backbone protein design methods for sequence library prediction in the therapeutic antibody Herceptin-HER2 interface.

Computational protein design methods can complement experimental screening and selection techniques by predicting libraries of low-energy sequences compatible with a desired structure and function. Incorporating backbone flexibility in computational design allows conformational adjustments that should broaden the range of predicted low-energy sequences. Here, we evaluate computational predictions of sequence libraries from different protocols for modeling backbone flexibility using the complex between the therapeutic antibody Herceptin and its target human epidermal growth factor receptor 2 (HER2) as a model system. Within the program RosettaDesign, three methods are compared: The first two use ensembles of structures generated by Monte Carlo protocols for near-native conformational sampling: kinematic closure (KIC) and backrub, and the third method uses snapshots from molecular dynamics (MD) simulations. KIC or backrub methods were better able to identify the amino acid residues experimentally observed by phage display in the Herceptin-HER2 interface than MD snapshots, which generated much larger conformational and sequence diversity. KIC and backrub, as well as fixed backbone simulations, captured the key mutation Asp98Trp in Herceptin, which leads to a further threefold affinity improvement of the already subnanomolar parental Herceptin-HER2 interface. Modeling subtle backbone conformational changes may assist in the design of sequence libraries for improving the affinity of antibody-antigen interfaces and could be suitable for other protein complexes for which structural information is available.