Dimerization Assay Research Articles

Novel receptor dimerization assay highlights a complex interplay between GT1b, SV2, and FGFRs

Novel receptor dimerization assay highlights a complex interplay between GT1b, SV2, and FGFRs

In vitro evaluation of dual-antigenic PV1 peptide vaccine in head and neck cancer patients

ABSTRACTPeptide vaccines derived from tumour-associated antigens have been used as an immunotherapeutic approach to induce specific cytotoxic immune response against tumour. We previously identified that MAGED4B and FJX1 proteins are overexpressed in HNSCC patients; and further demonstrated that two HLA-A2-restricted 9–11 amino acid peptides derived from these proteins were able to induce anti-tumour immune responses in vitro independently using PBMCs isolated from these patients. In this study, we evaluated the immunogenicity and efficacy of a dual-antigenic peptide vaccine (PV1), comprised of MAGED4B and FJX1 peptides in HNSCC patients. We first demonstrated that 94.8% of HNSCC patients expressed MAGED4B and/or FJX1 by immunohistochemistry, suggesting that PV1 could benefit the majority of HNSCC patients. The presence of pre-existing MAGED4B and FJX1-specific T-cells was detected using a HLA-A2 dimer assay and efficacy of PV1 to induce T-cell to secrete cytotoxic cytokine was evaluated using ELISPOT assay. Pre-existing PV1-specific T-cells were detected in all patients. Notably, we demonstrated that patients’ T-cells were able to secrete cytotoxic cytokines upon exposure to target cells expressing the respective antigen post PV1 stimulation. Furthermore, patients with high expression of MAGED4B and FJX1 in their tumours were more responsive to PV1 stimulation, demonstrating the specificity of the PV1 peptide vaccine. Additionally, we also demonstrated the expression of MAGED4B and FJX1 in breast, lung, colon, prostate and rectal cancer suggesting the potential use of PV1 in these cancers. In summary, PV1 could be a good vaccine candidate for the treatment of HNSCC patients and other cancers expressing these antigens.

2028 Discovery and evaluation of FOXP3 dimerization inhibitors

OBJECTIVES/SPECIFIC AIMS: Immuno-oncology (IO) strategies are promising new approaches for the treatment of a variety of malignancies, including multiple myeloma (MM). Regulatory T cells (Tregs), which suppress effector T cell function, are a limitation to durable IO responses. The transcription factor FOXP3 is critical for the mature Treg phenotype. FOXP3 homodimerization is required for DNA binding and transcriptional activity, and mutations mapping to the dimerization region are associated with IPEX syndrome, resulting in dysfunctional Tregs in humans. We therefore hypothesize that inhibitors of FOXP3 dimerization will repress Treg suppression and enhance the anti-MM activity of IO. METHODS/STUDY POPULATION: To discover FOXP3 dimerization inhibitors, we are modeling FOXP3 homodimerization in vitro. Currently, we are optimizing an ALPHA screen and an ELISA-based dimerization assay using recombinant full length and truncated versions of FOXP3 to discover peptidomimetics that inhibit homodimerization. Induced Tregs expanded from human PBMCs will be treated with lead biologics and functional assays will be performed. RESULTS/ANTICIPATED RESULTS: Here we demonstrate Treg suppression of T cell proliferation and IFN-γ secretion after 5 days of co-culture under basal conditions. Additionally, we developed a MM/T cell co-culture system to measure anti-MM T cell responses and show decreased anti-MM T cell activity in the presence of Tregs. We expect to exploit the assays outlined here to demonstrate defective Treg suppression when FOXP3 dimerization is inhibited. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies support drug discovery efforts that will ultimately improve IO therapies for patients with MM.

FRET analysis of HIV-1 Gag and GagPol interactions.

The Gag protein of HIV multimerizes to form viral particles. The GagPol protein encoding virus‐specific enzymes, such as protease, reverse transcriptase, and integrase, is incorporated into HIV particles via interactions with Gag. The catalytically active forms of these enzymes are dimeric or tetrameric. We employed Förster resonance energy transfer (FRET) assays to evaluate Gag–Gag, Gag–GagPol, and GagPol–GagPol interactions and investigated Gag and Pol interdomains tolerant to fluorescent protein insertion for FRET assays. Our data indicated that the matrix (MA)–capsid (CA) domain junction in the Gag region and the Gag C terminus were equally available for Gag–Gag and Gag–GagPol interaction assays. For GagPol dimerization assays, insertion at the MA–CA domain junction was most favorable.

Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides

The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β3-peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β3-peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes.

Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity.

Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity.IMPORTANCE Despite the availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that postexposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, the lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here we combined structure-guided mutagenesis with cellular, biochemical, and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provide evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher-order oligomers necessary to promote plasma membrane-budding activity.

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA‐binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re‐analyzing ChIP‐Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type‐specific POU factor function is determined by select residues that affect DNA‐dependent dimerization.

Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function

The level of the transcription factor Nanog directly determines the efficiency of mouse embryonic stem cell self-renewal. Nanog protein exists as a dimer with the dimerization domain composed of a simple repeat region in which every fifth residue is a tryptophan, the tryptophan repeat (WR). Although WR is necessary to enable Nanog to confer LIF-independent self-renewal, the mechanism of dimerization and the effect of modulating dimerization strength have been unclear. Here we couple mutagenesis with functional and dimerization assays to show that the number of tryptophans within the WR is linked to the strength of homodimerization, Sox2 heterodimerization and self-renewal activity. A reduction in the number of tryptophan residues leads initially to a gradual reduction in activity before a precipitous reduction in activity occurs upon reduction in tryptophan number below eight. Further functional attrition follows subsequent tryptophan number reduction with substitution of all tryptophan residues ablating dimerization and self-renewal function completely. A strong positional influence of tryptophans exists, with residues at the WR termini contributing more to Nanog function, particularly at the N-terminal end. Limited proteolysis demonstrates that a structural core of Nanog encompassing the homeodomain and the tryptophan repeat can support LIF-independent colony formation. These results increase understanding of the molecular interactions occurring between transcription factor subunits at the core of the pluripotency gene regulatory network and will enhance our ability to control pluripotent cell self-renewal and differentiation.

Development of a receptor dimerization assay to study BoNT/A and FGFR interactions

Development of a receptor dimerization assay to study BoNT/A and FGFR interactions

Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface

Spider dragline silk is a natural polymer harboring unique physical and biochemical properties that make it an ideal biomaterial. Artificial silk production requires an understanding of the in vivo mechanisms spiders use to convert soluble proteins, called spidroins, into insoluble fibers. Controlled dimerization of the spidroin N-terminal domain (NTD) is crucial to this process. Here, we report the crystal structure of the Nephila clavipes major ampullate spidroin NTD dimer. Comparison of our N. clavipes NTD structure with previously determined Euprosthenops australis NTD structures reveals subtle conformational alterations that lead to differences in how the subunits are arranged at the dimer interface. We observe a subset of contacts that are specific to each ortholog, as well as a substantial increase in asymmetry in the interactions observed at the N. clavipes NTD dimer interface. These asymmetric interactions include novel intermolecular salt bridges that provide new insights into the mechanism of NTD dimerization. We also observe a unique intramolecular "handshake" interaction between two conserved acidic residues that our data suggest adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization. The results of a panel of tryptophan fluorescence dimerization assays probing the importance of these interactions support our structural observations. Based on our findings, we propose that conformational selectivity and plasticity at the NTD dimer interface play a role in the pH-dependent transition of the NTD from monomer to stably associated dimer as the spidroin progresses through the silk extrusion duct.

Dimeric FcγR Ectodomains as Probes of the Fc Receptor Function of Anti-Influenza Virus IgG.

Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface-expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgG-opsonized influenza A hemagglutinin correlated with dimeric rsFcγRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcγR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants.

A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.

c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2′hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses.

Determining the CQC-Mediated Interactions in the Mucin 1 Homodimer

Mucin 1 (MUC1) is a highly glycosylated single pass membrane protein that serves as a selective barrier against pathogens and engages in signal transduction influencing cellular growth and differentiation. Overexpression of mucins have been linked to 80-90% of all human solid tissue cancers. Constitutive homodimerization, as a result of overexpression, leads to the formation of complexes with growth factor receptors and targeting to the nucleus, where MUC1 interacts with effector proteins regulating gene expression.Recent studies have shown that the MUC1 cytosolic membrane proximal CQC motif promotes dimerization under oxidizing conditions, suggesting that the motif may act as a redox switch in response to changes in oxidant levels. Preliminary studies have shown that the transmembrane domain (TMD) of MUC1 lacking the CQC motif form weak dimers, confirming their contribution to dimerization. Also, our results show that TMDs with the CQC motif dimerize strongly when the motif is in an oxidizing environment, further enforcing the role of the motif acts as a redox switch.Using the ToxR dimerization assay, the effects of single amino acid substitutions in TMDs with the CQC juxtamembrane area are analyzed. With these substitutions, the dimer interface can be elucidated. Our goal is to determine (1) whether certain amino acids in the TMD play a key role in forming MUC1 homodimers, and (2) whether the CQC motif induces a structural change when it functions as a redox switch. These conclusions will contribute to our understanding of MUC1 dimerization and provide insights for the development of novel therapeutic strategies.

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain

BackgroundSmall molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified. MethodsThree peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the KD of the peptides towards the C-terminal domain of Hsp90. ResultsMD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC50 values of 2.08μM for peptide H2 and 8.96μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a KD of 1.29nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a KD of 1.02μM and 1.46μM, respectively. ConclusionsThese results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity. General significanceThese results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.

Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR.

The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate invitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.

ID: 145: Theiler’s virus L∗ protein inhibits RNase L dimerization and activation by binding to the 2-5A-binding pocket of the enzyme

We observed previously that the L∗ protein of Theiler’s virus (picornavirus) inhibited RNase L, through direct protein–protein interaction. Interestingly, RNase L inhibition by L∗ exhibits species-specificity as L∗ inhibits mouse but not human RNase L [1] . We took advantage of this species specificity to map the L∗ binding site on RNase L, by testing L∗-mediated inhibition of a series of human-mouse RNase L chimeras. Our results suggest that L∗ interacts at two sites within RNase L ankyrin repeats 1 and 2. According to the recently solved structure of dimeric RNase L [2] , [3] , our results are compatible with RNase L inhibition through both dimerization and 2-5A-binding antagonism. We therefore established a crosslinking-based RNase L dimerization assay and show that L∗ indeed inhibits RNase L dimerization. Next, we analyzed whether L∗ could interfere with 2-5A binding. Competitive binding studies by surface plasmon resonance demonstrated that L∗ indeed competes with 2-5A binding to mouse but not human RNase L. In conclusion, protein L∗ of Theiler’s virus inhibits the OAS/RNase L pathway by binding to RNase L ankyrin repeats 1 and 2, thereby preventing 2-5A-mediated dimerization and activation of RNase L. L∗ is the first viral protein we know of that antagonizes the OAS/RNase L pathway by acting on the effector enzyme.

Elucidating the structural consequences of CQC‐mediated dimerization of MUC1

Mucin 1 (MUC1) is a highly glycosylated single pass membrane protein that serves as a selective barrier against pathogens and engages in signal transduction influencing cellular growth and differentiation. Overexpression of mucins have been linked to 80‐90% of all human solid tissue cancers. Constitutive homodimerization, as a result of overexpression, leads to the formation of complexes with growth factor receptors and targeting to the nucleus, where MUC1 interacts with effector proteins regulating gene expression.Recent studies have shown that the MUC1 cytosolic membrane proximal CQC motif promotes dimerization under oxidizing conditions, suggesting that the motif may act as a redox switch in response to changes of oxidant levels. Preliminary studies have shown that the transmembrane domain (TMD) of MUC1 lacking the CQC motif form weak dimers, confirming their contribution to dimerization. Also, results show that TMDs containing the CQC in an oxidizing environment exhibit increased dimerization, further enforcing the role of the motif acts as a redox switch.Using the ToxR dimerization assay, the effects of single amino acid substitutions in TMDs with the CQC motif and TMDs with the CQC‐>AQA motif are being measured. Our goal is to determine (1) whether certain amino acids in the TMD play a key role in forming MUC1 homodimers, and (2) whether the CQC motif induces a structural change when it functions as a redox switch. These conclusions will contribute to our understanding of MUC1 dimerization and provide insights for the development of novel therapeutic strategies.

Predicting the risk of recurrent venous thromboembolism (VTE).

To optimize patient outcomes with anticoagulation for venous thromboembolism (VTE), it is essential to assess patients for their recurrence risk. In this article, I will review the impact of clinical and laboratory risk factors for recurrent VTE. The presence or absence of VTE risk factors at the time of the index thrombotic event provides important information regarding recurrence risk. Patients with potent situational risk factors for thrombosis (e.g., surgery) are at low risk for recurrence while patients suffering unprovoked events are at high risk for recurrence. The presence of non-surgical clinical risk factors place patients at intermediate recurrence risk. Other clinical risk factors of variable recurrence potential include age, sex, cancer, pregnancy/puerperium, hormonal therapy and obesity. Laboratory risk factors for recurrence include thrombophilia, D dimer and other global coagulation assays as well as residual venous obstruction. Several multivariate VTE risk assessment models have been developed that combine clinical and laboratory risk factors of recurrence. If validated, these risk scores may allow for personalized anticoagulation therapy tailored to patients' individual recurrence risk.

Emery-Dreifuss muscular dystrophy mutations impair TRC40-mediated targeting of emerin to the inner nuclear membrane.

Emerin is a tail-anchored protein that is found predominantly at the inner nuclear membrane (INM), where it associates with components of the nuclear lamina. Mutations in the emerin gene cause Emery-Dreifuss muscular dystrophy (EDMD), an X-linked recessive disease. Here, we report that the TRC40/GET pathway for post-translational insertion of tail-anchored proteins into membranes is involved in emerin-trafficking. Using proximity ligation assays, we show that emerin interacts with TRC40 in situ. Emerin expressed in bacteria or in a cell-free lysate was inserted into microsomal membranes in an ATP- and TRC40-dependent manner. Dominant-negative fragments of the TRC40-receptor proteins WRB and CAML (also known as CAMLG) inhibited membrane insertion. A rapamycin-based dimerization assay revealed correct transport of wild-type emerin to the INM, whereas TRC40-binding, membrane integration and INM-targeting of emerin mutant proteins that occur in EDMD was disturbed. Our results suggest that the mode of membrane integration contributes to correct targeting of emerin to the INM.