SH2 Protein Research Articles

Profiling human Src homology 2 (SH2) domain proteins and ligand discovery using a peptide-hybrid small molecule microarray

A 396-member peptide-hybrid small molecule microarray was fabricated to profile small molecule ligands across 15 SH2 proteins, revealing hits against Lck and Grb2.

High Affinity Neurexin Binding to Cell Adhesion G-protein-coupled Receptor CIRL1/Latrophilin-1 Produces an Intercellular Adhesion Complex

The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

JAK's SOCS: A Mechanism of Inhibition

SOCS1 and SOCS3 are specific inhibitors for JAK tyrosine kinases. In this issue of Immunity, Babon et al. (2012) discovered the inhibition mechanism of SOCS3 by employing nuclear magnetic resonance and classical enzyme kinetics.

LNCaP prostate cancer cells with autocrine interleukin‐6 expression are resistant to IL‐6‐induced neuroendocrine differentiation due to increased expression of suppressors of cytokine signaling

Neuroendocrine differentiation (NED) is one of the mechanisms underlying development of castration-resistant prostate cancer (CRPC). In this study, we investigated IL-6-induced NED in two LNCaP sublines. LNCaP-S17, an LNCaP subline that secretes IL-6, and LNCaP-C3, a control subline that does not express IL-6, were analyzed for IL-6-induced NED, activation of JAK2 and STAT3 pathways, and expression of IL-6/IL-6R signaling proteins and downstream target genes. IL-6 did not induce NED in LNCaP-S17 cells, even though IL-6 induced NED in LNCaP-C3 cells. IL-6 activated JAK2 and STAT3 pathways in LNCaP-C3 cells but not in LNCaP-S17 cells. IL-6 did not activate ERK1/2, AKT, or NF-κB pathways in either cell line. Both LNCaP-C3 and LNCaP-S17 cell lines expressed IL-6R, gp130, and TYK2 at almost the same levels and did not express JAK1 or JAK3. The basal level of JAK2 expression was slightly higher in LNCaP-C3 cells than in LNCaP-S17 cells. Two suppressors of cytokine signaling, SOCS7 and cytokine-inducible SH2 protein (CIS), were expressed constitutively at higher levels in LNCaP-S17 cells than in LNCaP-C3 cells, while SOCS1 to SOCS6 were expressed at approximately the same levels. Using siRNA to knockdown SOCS7 and CIS expression in LNCaP-S17 cells led to increased phosphorylation of STAT3 upon IL-6 stimulation. LNCaP-S17 cells are resistant to exogenous IL-6-induced NED due to increased levels of CIS/SOCS7 that block activation of JAK2-STAT3 pathways.

The Gads Adaptor Protein Is Required to Mediate Lymphoid Disease Downstream of BCR-ABL,

Abstract 3750Chronic Myeloid Leukemia is mediated by the BCR-ABL chromosomal translocation. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates a robust myeloproliferative disease that is fatal in recipient animals within 21–28 days that have received transduced marrow from donor mice primed with 5-fluorouracil. Structure/function experiments revealed that mice receiving bone marrow expressing BCR-ABL Y177F generate a T cell lymphoma with a latency of 73–116 days, demonstrating that the SH2 proteins that bind to BCR-ABL pY177 are critical for promoting myeloproliferative disease.There are three members of the Grb2 adaptor protein family: Grb2, Gads and Grap. Biochemical experiments suggest that the Grb2 adaptor protein binds to BCR-ABL Y177 and acts to couple BCR-ABL to the Gab2 adaptor protein. In turn, Gab2 links to activation of the Ras and PI 3 kinase signal transduction pathways. Previous studies from our group revealed that Gads binds to BCR-ABL in vitro. We hypothesized that Gads plays an important role downstream of BCR-ABL. To test this objective, we performed retroviral transduction-bone marrow transplantation experiments utilizing bone marrow from wild type and Gads-deficient mice.Thymocytes from Gads−/− mice exhibit defective proliferation in response to CD3 activation and deficits in positive and negative selection. The loss of Gads results uncoupling of Slp-76 and Lat, and impaired PLCγ phosphorylation in thymocytes. No other prominent phenotypes have been documented in this animal model.We show that retroviral transduction-bone marrow transplantation experiments completed with 5-fluoruracil primed bone marrow display a similar latency in BCR-ABL expressing bone marrow from wild type or Gads-deficient mice.Retroviral transduction-bone marrow transplantation experiments can also be completed with normal (unprimed) bone marrow which generates a mixed syndrome comprising myeloproliferative disease, B cell Acute Lymphoid Leukemia and macrophage tumours with a latency from 30–70 days in mice transduced with BCR-ABL. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads−/− bone marrow expressing BCR-ABL and all mice succumb to myeloproliferative disease at 21 days. These data suggest that Gads is required for lymphoproliferative disease in this model.We have examined signaling events in the CML-T1 and LAMA-84 CML cell lines that co-express GRB2 and GADS in order to develop models to account for our observations. GRB2 and GADS both co-immunoprecipitate BCR-ABL in both cell lines. However, there is remarkable specificity in the complexes that bind to each adaptor protein. For example, GRB2 co-immunoprecipitates the SHC adaptor protein (but not GAB2), whereas GADS co-immunoprecipitates GAB2 and SLP-76 from both CML cell lines. We are currently performing experiments to confirm these observations in BCR-ABL expressing Acute Lymphoid Leukemia patient specimens.Our data suggests that BCR-ABL mediates Y177-dependent signaling via Grb2-dependent activation of Ras and PI 3 kinase in a myeloproliferative model of CML. However, in keeping with the important role that Gads plays in maintaining lymphocyte homeostasis, Gads mediates lymphoproliferative disease downstream of BCR-ABL, through the recruitment of specific signaling intermediates. Disclosures:No relevant conflicts of interest to declare.

MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo

Abstract 2181Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s.Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency.We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway.In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation. Disclosures:No relevant conflicts of interest to declare.

Suppressor of Cytokine Signaling-1 (SOCS1) Inhibits Lymphocyte Recruitment into the Retina and Protects SOCS1 Transgenic Rats and Mice from Ocular Inflammation

Suppressors of cytokine signaling (SOCS) proteins regulate the intensity and duration of cytokine signals and defective expression of SOCS1 and SOCS3 has been reported in a number of human diseases. The purpose of this study was to investigate the role of SOCS1 in intraocular inflammatory diseases (uveitis) and whether SOCS1 expression is defective in patients with ocular inflammatory diseases. Blood from patients with scleritis or healthy human volunteers was analyzed for SOCS expression by RNase protection assay and RT-PCR. The authors generated SOCS1 transgenic rats and mice (SOCS1-Tg), induced experimental autoimmune uveoretinitis (EAU) by active immunization with interphotoreceptor retinal binding protein or adoptive transfer of uveitogenic T cells, and investigated effects of SOCS1 overexpression on EAU. SOCS1-mediated protection of retinal cells from apoptosis was assessed by annexin V staining. Induction of cytokine-induced SH2 protein was comparable between patients and volunteers, whereas 80% of lymphocytes from patients with scleritis failed to induce SOCS1 in response to IL-2. Compared with wild-type littermates, SOCS1-Tg rats/mice developed less severe EAU. Constitutive overexpression of SOCS1 in retina inhibited expression of chemokines (CCL17, CCL20, CXCL9, CXCL10), reduced Th17/Th1 expansion, and inhibited recruitment of inflammatory cells into the retina. The authors also show that SOCS1 protected retinal cells from staurosporine as well as H₂O₂-induced apoptosis. Defective expression of SOCS1 in patients with scleritis, taken together with SOCS1-mediated protection of neuroretinal cells from apoptosis, suggest that SOCS1 has neuroprotective function in the retina, implying that administration of SOCS1 mimetic peptides may be useful in treating uveitis or scleritis.

SLAM Family Receptors and SAP Adaptors in Immunity

The signaling lymphocyte activation molecule (SLAM)-associated protein, SAP, was first identified as the protein affected in most cases of X-linked lymphoproliferative (XLP) syndrome, a rare genetic disorder characterized by abnormal responses to Epstein-Barr virus infection, lymphoproliferative syndromes, and dysgammaglobulinemia. SAP consists almost entirely of a single SH2 protein domain that interacts with the cytoplasmic tail of SLAM and related receptors, including 2B4, Ly108, CD84, Ly9, and potentially CRACC. SLAM family members are now recognized as important immunomodulatory receptors with roles in cytotoxicity, humoral immunity, autoimmunity, cell survival, lymphocyte development, and cell adhesion. In this review, we cover recent findings on the roles of SLAM family receptors and the SAP family of adaptors, with a focus on their regulation of the pathways involved in the pathogenesis of XLP and other immune disorders.

Comparison of the Frequency of Functional SH3 Domains with Different Limited Sets of Amino Acids Using mRNA Display

Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory.

The WD40-Repeats of Ahi-1 Oncogene Critical for Protein Interaction Between Ahi-1 and BCR-ABL Modulates Imatinib-Induced Apoptosis In BCR-ABL-Transduced Cells

AbstractAbstract 4181Ahi-1 (Abelson helper integration site-1) is an oncogene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma. The Ahi-1/AHI-1 protein contains an SH3 domain, multiple SH3 binding sites and a WD40-repeat domain, all known to be important mediators of protein-protein interactions. Human AHI-1 is highly deregulated in human leukemic cells, particularly in BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML). We have demonstrated that overexpression of Ahi-1 in primitive hematopoietic cells confers a growth advantage in vitro and induces leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that plays a major role in the genesis of CML. AHI-1 can physically interact with BCR-ABL and JAK2 in CML cells and this interaction complex further mediates tyrosine kinase inhibitor (TKI) response/resistance of CML stem/progenitor cells. Despite its importance, the mechanism by which this complex affects cell proliferation and survival and regulates sensitivity of CML cells to TKIs remains unknown. To identify and characterize which functional domain(s) of Ahi-1 is critical for its interaction with BCR-ABL and/or Jak2, full length Ahi-1 and several mutant forms, including N-terminal deletion (N-terΔ, containing both SH3 and WD40-repeat domains), SH3 deletion (SH3Δ) and double WD40-repeat domain and SH3 domain deletions (SH3WD40Δ) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Epitope-tagged full length and mutant Ahi-1 constructs were also transiently expressed in 293T cells co-expressed with either BCR-ABL or Jak2. Co-IP experiments showed that Ahi-1 is highly expressed and stably associated with BCR-ABL-Jak2 complex in BCR-ABL inducible cells co-transduced with full-length Ahi-1 and that it can directly interact with Jak2; Ahi-1 was detected in both Ahi-1-transduced BaF3 cells (without BCR-ABL) and BCR-ABL inducible cells after IP with a Jak2 antibody. Interestingly, N-terminal region of Ahi-1 was required for Ahi-1-Jak2 interaction as a predicted 70 kD product was not detectable in the same cells transduced with the Ahi-1N-terΔ mutant. In contrast, N-terminal region of Ahi-1 was not associated with Ahi-1-BCR-ABL interaction since BCR-ABL was still detectable in BCR-ABL inducible cells co-transduced with the Ahi-1N-terΔ mutant after IP with the anti-ABL antibody. The deletion of either the SH3 domain or both the SH3 and WD40-repeat domains did not interfere with their interaction with Jak2. Ahi-1 lacking both the SH3 and WD40-repeat domains lost its ability to interact with BCR-ABL when the SH3WD40Δ mutant and BCR-ABL were co-expressed in 293T cells and the SH3 deletion did not affect Ahi-1's interaction with BCR-ABL, indicating that the WD40-repeart domain is required for a direct Ahi-1-BCR-ABL interaction. As expected, overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells after imatinib (IM) treatment compared to BCR-ABL inducible cells (5.9% and 8% v.s.26% and 34% with either 2 or 5 μM IM) after 24 hours. Interestingly, cells expressing the SH3WD40Δ mutant displayed dramatically increased cell sensitivity to IM with increased Annexin V+ cells (62% and 69% v.s.5.9% and 8%), while cells expressing the SH3Δ and the N-terΔ mutants had similar numbers of Annexin V+ cells as compared to BCR-ABL inducible cells (32% and 16% v.s.26% with 2 μM IM). Colony forming cell assays (CFC) further showed significantly reduced growth factor independent CFC numbers generated from the SH3WD40Δ mutant compared to BCR-ABL inducible cells co-expressed with the full-length Ahi-1 (4% v.s.46% with 5μM IM compared to untreated cells). Interestingly, a gene expression study comparing full-length Ahi-1-transduced BCR-ABL cells with its mutants further demonstrated that these changes were associated with deregulated expression of Bcl-2 and Cis (Cytokine inducible SH2 protein) involved in apoptosis. These results suggest that the WD40-repeat domain is important for Ahi-1's role in mediating IM-induced apoptosis and that targeting this critical domain of Ahi-1may provide a novel therapeutic option for treatment of CML. Disclosures:No relevant conflicts of interest to declare.

Knocking off the suppressors of cytokine signaling (SOCS): their roles in mammalian pregnancy

This review discusses the possible role of the suppressor of cytokine signaling (SOCS) proteins in mammalian reproduction. SOCS are regulatory proteins that are rapidly transcribed in response to intracellular Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling, a cascade governing biological functions including cytokine-induced immunological responses and reproductive processes. For instance STAT3 appears to mediate trophoblast invasion induced by LIF. The SOCS family includes 8 members (cytokine-inducible SH2 protein [CIS] and SOCS1-7) that orchestrate distinct reactions by antagonizing STAT activation. Emerging evidence points to a role of some family members in synchronizing Th1/Th2 cell profiles, the balance in which is considered vital to pregnancy maintenance. The reproductive phenotypes of mutant mice harboring targeted disruption of SOCS gene isoforms offer insights for reproductive immunology, trophoblast function and human pregnancy. CIS transgenic mice display impaired responses to IL-2 and resemble STAT5 deficient mice, except they are fertile. SOCS1 deficiency leads to an overabundance of IFNgamma signaling, yet SOCS1 null mutant mice are able to reproduce. Lack of SOCS3 is embryonically lethal due to placental insufficiency, while SOCS3 over-expression leads to elevated Th2 responses. SOCS3 seems to be vital for reproduction by regulating LIF-driven trophoblast differentiation. SOCS5 inhibits IL-4 signaling, yet the SOCS5 transgenic mouse has no conspicuous reproductive phenotype. SOCS-6 and SOCS-7 null mutant mice display growth retardation. In summary, SOCS proteins are avidly involved in fine regulation of immunological and other vital cellular responses. Many of the above phenotypes present contradictions to accepted reproductive immunological paradigms.

Identification of SH3 domain interaction partners of human FasL (CD178) by phage display screening

Background -Fas ligand is a cytotoxic effector molecule of T and NK cells which is characterized by an intracellular N-terminal polyproline region that serves as a docking site for SH3 and WW domain proteins. Several previously described Fas ligand-interacting SH3 domain proteins turned out to be crucial for the regulation of storage, expression and function of the death factor. Recent observations, however, indicate that Fas ligand is also subject to posttranslational modifications including shedding and intramembrane proteolysis. This results in the generation of short intracellular fragments that might either be degraded or translocate to the nucleus to influence transcription. So far, protein-protein interactions that specifically regulate the fate of the intracellular fragments have not been identified.Results -In order to further define the SH3 domain interactome of the intracellular region of Fas ligand, we now screened a human SH3 domain phage display library. In addition to known SH3 domains mediating binding to the Fas ligand proline-rich domain, we were able to identify a number of additional SH3 domains that might also associate with FasL. Potential functional implications of the new binding proteins for the death factor's biology are discussed. For Tec kinases and sorting nexins, the observed interactions were verified in cellular systems by pulldown experiments.Conclusion -We provide an extended list of putative Fas ligand interaction partners, confirming previously identified interactions, but also introducing several novel SH3 domain proteins that might be important regulators of Fas ligand function.

The Adapter Protein SLP-76 Mediates “Outside-In” Integrin Signaling and Function in T Cells

The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

Age-Specific Morphofunctional Changes in Cambial Cells and Their Derivatives in Human Skin

The count of cambial cells decreases with age in the total morphofunctional zone of the skin. However, their number determines the stretching of the daughter cells, which, in turn, activates the Src domain of protein SH2. Twelve cambial cells is the optimal quantity, at which the SH2 domain (responsible later for full-value stretching and looping of chromosomes near telomeres in the region of epithelial cells formation) is sufficiently active, which is characteristic of the age of 20-40 and 41-59 years. If the number of cambial cells is less than 12, stretching of the nuclei and expression of SH2 domain reduce, this leading to looping of lesser sites of chromosomes, modulating the transcription; the expression of Src and RhoA proteins is imbalanced, which leads to reduction of cell proliferation, modification of the direction of the cell's basic axis, and to hyperdesquamation. This is characteristic of the age of over 60 years. After 75 years of age the number of cambial cells in both subunits of the epidermal basal layer decreased to 7 and is close to the threshold level (6 cells), when interactions between fields of the two subunits are weak or zero. Therefore, despite the involvement of both subunits (14 cells), cell stretching and expression of the Src protein SH2 domain in them drop sharply, while activity of RhoA protein increased, which leads to looping of chromosomes closer to the centromeres, that is, to active development of fibroblasts, which is characteristic of subjects over 75 years of age.

A Collective Variable for the Efficient Exploration of Protein Beta-Sheet Structures: Application to SH3 and GB1.

We introduce a new class of collective variables which allow forming efficiently beta-sheet structures in all-atom explicit-solvent simulations of proteins. By this approach we are able to systematically fold a 16-residue beta hairpin using metadynamics on a single replica. Application to the 56-residue SH3 and GB1 proteins show that, starting from extended states, in ∼100 ns tens of structures containing more than 30% beta-sheet are obtained, including parts of the native fold. Using these variables may allow folding moderate size proteins with an accurate explicit solvent description. Moreover, it may allow investigating the presence of misfolded states that are relevant for diseases (e.g., prion and Alzheimer) and studying beta-aggregation (amyloid diseases).

Computational protein design as a tool for fold recognition

Computationally designed protein sequences have been proposed as a basis to perform fold recognition and homology searching. To investigate this possibility, an automated procedure is used to completely redesign 24 SH3 proteins and 22 SH2 proteins. We use the experimental backbone coordinates as fixed templates in the folded state and a molecular mechanics model to compute the pairwise interaction energies between all sidechain types and conformations. Energy calculations are done with the Proteins@Home volunteer computing platform. A heuristic algorithm is then used to scan the sequence and conformational space for optimal solutions. We produced 200,000-450,000 sequences for each backbone template. The designed sequences ressemble moderately-distant, natural homologues of the initial templates, according to their identity scores and their similarity with respect to the Pfam sets of SH2 and SH3 domains. Standard homology detection tools document their native-like character: the Conserved Domain Database recognizes 61% (52%) of our low-energy sequences as SH3 (SH2) domains; the SUPERFAMILY, Hidden-Markov Model library recognizes 81% (84%). Conversely, position specific scoring matrices (PSSMs) derived from our designed sequences can be used to detect natural homologues in sequence databases. Within SwissProt, a set of natural SH3 PSSMs detects 772 SH3 domains, for example; our designed PSSMs detect 67% of these, plus one additional sequence and two false positives. If six amino acids involved in substrate binding (a selective pressure not accounted for in our design) are reset to their experimental types, then 77% of the experimental SH3 domains are detected. Results for the SH2 domains are similar. Several directions to improve the method further are discussed.

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.

The XLP syndrome protein SAP interacts with SH3 proteins to regulate T cell signaling and proliferation

The gene sap/shd1a, which encodes a 128-residue SH2 domain protein, is frequently deleted or mutated in the X-linked lymphoproliferative syndrome (XLP). The SAP SH2 domain differs from others in the same class in that it is not only capable of binding to a phosphotyrosine-containing peptide, it can also associate with an SH3 domain using a distinct surface. This novel mode of ligand-binding is initially discovered in the SLAM-SAP-Fyn complex that plays a critical role in T cell and natural killer cell activation. To identify additional binding partners for SAP, we screened a panel of 12 SH3 domains derived from regulatory proteins and identified NCK1 as a novel target of SAP in T cells. NMR analysis demonstrated that the NCK1 and Fyn SH3 domains possessed comparable affinities for SAP and engaged the same set of residues on the surface of the SAP SH2 domain. Depletion of SAP by siRNA caused a significant decrease in NCK1 tyrosine phosphorylation as well as the phosphorylation of other T cell receptor (TCR) downstream proteins such as LAT and SLP-76. Moreover, SAP was shown to regulate T cell proliferation through the MAP-kinase Erk. Taken together, our work identifies NCK1 as a novel physiological partner for SAP and a direct regulator of TCR signaling and T cell proliferation.

The analysis of CIS, SOCS1, SOSC2 and SOCS3 transcript levels in peripheral blood mononuclear cells of systemic lupus erythematosus and rheumatoid arthritis patients

Being expressed in immune cells, cytokine-inducible SH2 protein (CIS) and suppressors of cytokine signaling proteins, SOCS1, SOCS2 and SOCS3, can regulate cytokine signaling and immune responses. To evaluate the possible expressional dysregulation of CIS, SOCS1, SOCS2 and SOCS3 in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients, the transcript levels of these genes in peripheral blood mononuclear cells (PBMCs) from SLE and RA patients were determined and statistically compared with those in PBMCs from normal individuals. It was found that SLE patients with the active disease activity significantly express higher CIS transcript levels than normal individuals and SLE patients with the inactive disease activity, whereas the difference in SOCS1, SOCS2 and SOCS3 transcript levels between normal individuals and SLE patients is not statistically significant. However, transcript levels of these CIS/SOCS genes in RA patients were not significantly different from those in normal individuals, except that treatment with a TNF-alpha-blocking agent in RA patients appears to enhance the CIS transcript expression, but down-regulates the SOCS2 transcript expression in PBMCs. These data suggest that CIS can serve as an SLE disease marker and may be involved in the pathogenesis of SLE, and that TNF-alpha may play an important role in the regulation of CIS and SOCS2 gene expression in PBMCs in vivo.

Multifunctional SH2 Domains

Crystal structures reveal how SH2 protein interaction domains can both enhance and inhibit activity of cytoplasmic tyrosine kinases.