Dimerization Capacity Research Articles

Morphological effect on Core@shell AuAg nanoparticles for detecting p‐aminothiophenol dimerization by surface‐enhanced Raman spectroscopy

AbstractA series of non‐spherical metallic Au and bimetallic core@shell AuAg nanoparticles (NPs) have been synthesized for SERS improvements. The bimetallic core@shell AuAg NPs were obtained through a controlled overgrowth of an Ag shell onto the surface of two types of non‐spherical Au NPs, used as seeds, Au nanooctaheda (Au NOc) and Au nanotriangles (Au NTs). This Ag overgrowth was able to produce bimetallic core@shell structures such as nanocubes and nanopyramids, respectively. Transmission electron microscopy (TEM) was used to determine the particle size and particle morphology for metallic and bimetallic NPs, and energy‐dispersive X‐ray (EDX) elemental mapping analysis confirmed the core@shell structure of the bimetallic NPs. The plasmonic absorption bands exhibited for each nanosystem were observed by UV–vis spectroscopy. The concentration of Au and Ag in the bimetallic systems was determined by inductively coupled plasma mass spectrometry (ICP‐MS). After synthesis and characterization, p‐aminothiophenol (PATP) was used as a model analyte to investigate the surface‐enhanced Raman spectroscopy (SERS) capabilities of the synthesized metallic and bimetallic nanosystems. In PATP, a dimerization reaction to 4,4′‐dimercaptoazobenzene (DMAB) is produced when it is adsorbed onto the surface of certain noble metals. This SERS analysis was performed at 10−4 M of PATP and by using two different laser wavelengths (532 and 785 nm) in all cases. In this context, we were able to detect the dimerization reaction of PATP to DMAB only for the bimetallic structures and under the 532 nm laser line. Moreover, we have found that the dimerization capacity also depends on the nanoparticle morphology.

Abstract LB_B04: Deciphering FGFR3-TACC3 oncogenic fusions

Abstract Chromosomal rearrangements of the fibroblast growth factor receptor (FGFR) genes that give rise to fusions are one of several mechanisms by which the FGF/FGFR signaling axis can become deregulated and result in enhanced signaling in cancer. We have previously identified truncation of exon (E) 18 of FGFR2 as a potent single-driver alteration in cancer, independently of the rearrangement (RE) partner. In contrast, the same does not appear to hold true for its E18-truncated ortholog FGFR3. We mined human oncogenomic datasets from Hartwig Medical Foundation (>2,500 WGS profiles) and Foundation Medicine (>200,000 hybrid-capture panel-seq profiles) for alterations affecting FGFR3. Examination of the structural variants affecting FGFR3 uncovered that 85% of all E18-truncating FGFR3 REs involved transforming acidic coiled-coil-containing protein 3 (TACC3) as the downstream fusion partner gene, while REs with intergenic regions and other known protein-encoding genes were much less frequent. Additionally, there was a clear predominance of self-interacting domains (95%) among all FGFR3 RE partners, with the coiled-coil domain being the most recurrent, suggesting enhanced receptor dimerization and downstream signaling capacity for the majority of the FGFR3 fusions. Our findings led to the generation of a compendium of FGFR3 structural variants which we then functionally tested. In vitro testing of mouse mammary epithelial cells expressing Fgfr3ΔE18-Tacc3E7-E16 fusion variants showed that both E18-truncation and a fusion partner were required for 3D outgrowth and signaling induction. In vivo evaluation of the oncogenic capacity of Fgfr3 variants using somatic delivery of lentiviruses to the mouse mammary gland via intraductal injection and lung via intratracheal injection found that Fgfr3ΔE18-Tacc3E7-E16 fusion variants rapidly induced mammary and lung tumor formation in wild-type and Wap-Cre;Cdh1F/F, and in wild-type and Trp53F/F mice, respectively. In contrast, Fgfr3full-length(FL), Fgfr3ΔE18 and Fgfr3FL–Tacc3E7-E16 were non-tumorigenic. Noteworthy, mammary tumors driven by Fgfr3ΔE18-Tacc3E7-E16 fusions were sensitive to the FGFR inhibitor AZD4547 during a drug intervention study. In summary, our findings show that E18-truncating FGFR3 alterations are recurrent across human cancers and, as opposed to FGFR2 E18-truncating alterations, FGFR3 appears to depend both on E18-truncation and an additional fusion partner with dimerizing capacity. Notably, TACC3 is the most recurrent 3’ fusion partner across all FGFR3 E18-truncating alterations, and somatic tumor modeling has shown that Fgfr3ΔE18-Tacc3 fusions drive mouse mammary and lung tumorigenesis, and are sensitive to FGFR targeted therapy. Citation Format: Julia Yemelyanenko, Jinhyuk Bhin, Sjoerd Klarenbeek, Ji-Ying Song, Catrin Lutz, Marieke van de Ven, Shridar Ganesan, Lodewyk F. A. Wessels, Daniel Zingg, Jos Jonkers. Deciphering FGFR3-TACC3 oncogenic fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B04.

Functional Dimerization of Serotonin Receptors: Role in Health and Depressive Disorders.

Understanding the neurobiological underpinnings of depressive disorder constitutes a pressing challenge in the fields of psychiatry and neurobiology. Depression represents one of the most prevalent forms of mental and behavioral disorders globally. Alterations in dimerization capacity can influence the functional characteristics of serotonin receptors and may constitute a contributing factor to the onset of depressive disorders. The objective of this review is to consolidate the current understanding of interactions within the 5-HT receptor family and between 5-HT receptors and members of other receptor families. Furthermore, it aims to elucidate the role of such complexes in depressive disorders and delineate the mechanisms through which antidepressants exert their effects.

Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B.

Purine-rich element binding protein B (Purβ) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purβ with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purβ. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purβ point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purβ. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purβ for ssDNA and diminished the interaction of Purβ with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purβ to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purβ.

Mutations in the α4-α5 allosteric lobe of RAS do not significantly impair RAS signaling or self-association

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

SPOP mutations promote p62/SQSTM1-dependent autophagy and Nrf2 activation in prostate cancer.

p62/SQSTM1 is a selective autophagy receptor that drives ubiquitinated cargos towards autophagic degradation. This receptor is also a stress-induced scaffold protein that helps cells to cope with oxidative stress through activation of the Nrf2 pathway. Functional disorders of p62 are closely associated with multiple neurodegenerative diseases and cancers. The gene encoding the E3 ubiquitin ligase substrate-binding adapter SPOP is frequently mutated in prostate cancer (PCa), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis remain poorly understood. Here, we report that cytoplasmic SPOP binds and induces the non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This protein modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing p62-dependent autophagy. Moreover, we show that SPOP relieves p62-mediated Keap1 sequestration, which ultimately decreases Nrf2-mediated transcriptional activation of antioxidant genes. We further show that PCa-associated SPOP mutants lose the capacity to ubiquitinate p62 and instead promote autophagy and the redox response in a dominant-negative manner. Thus, our findings indicate oncogenic roles of autophagy and Nrf2 activation in the tumorigenesis of SPOP-mutated PCa.

The Complex Formed by Group I Metabotropic Glutamate Receptor (mGluR) and Homer1a Plays a Central Role in Metaplasticity and Homeostatic Synaptic Scaling.

G-protein-coupled receptors can be constitutively activated following physical interaction with intracellular proteins. The first example described was the constitutive activation of Group I metabotropic glutamate receptors (mGluR: mGluR1,5) following their interaction with Homer1a, an activity-inducible early-termination variant of the scaffolding protein Homer that lacks dimerization capacity (Ango et al., 2001). Homer1a disrupts the links, maintained by the long form of Homer (cross-linking Homers), between mGluR1,5 and the Shank-GKAP-PSD-95-ionotropic glutamate receptor network. Two characteristics of the constitutive activation of the Group I mGluR-Homer1a complex are particularly interesting: (1) it affects a large number of synapses in which Homer1a is upregulated following enhanced, long-lasting neuronal activity; and (2) it mainly depends on Homer1a protein turnover. The constitutively active Group I mGluR-Homer1a complex is involved in the two main forms of non-Hebbian neuronal plasticity: "metaplasticity" and "homeostatic synaptic scaling," which are implicated in a large series of physiological and pathologic processes. Those include non-Hebbian plasticity observed in visual system, synapses modulated by addictive drugs (rewarded synapses), chronically overactivated synaptic networks, normal sleep, and sleep deprivation.

A variant in human AIOLOS impairs adaptive immunity by interfering with IKAROS.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

Curtailing FGF19’s mitogenicity by suppressing its receptor dimerization ability

As a physiological regulator of bile acid homeostasis, FGF19 is also a potent insulin sensitizer capable of normalizing plasma glucose concentration, improving lipid profile, ameliorating fatty liver disease, and causing weight loss in both diabetic and diet-induced obesity mice. There is therefore a major interest in developing FGF19 as a therapeutic agent for treating type 2 diabetes and cholestatic liver disease. However, the known tumorigenic risk associated with prolonged FGF19 administration is a major hurdle in realizing its clinical potential. Here, we show that nonmitogenic FGF19 variants that retain the full beneficial glucose-lowering and bile acid regulatory activities of WT FGF19 (FGF19WT) can be engineered by diminishing FGF19's ability to induce dimerization of its cognate FGF receptors (FGFR). As proof of principle, we generated three such variants, each with a partial defect in binding affinity to FGFR (FGF19ΔFGFR) and its coreceptors, i.e., βklotho (FGF19ΔKLB) or heparan sulfate (FGF19ΔHBS). Pharmacological assays in WT and db/db mice confirmed that these variants incur a dramatic loss in mitogenic activity, yet are indistinguishable from FGF19WT in eliciting glycemic control and regulating bile acid synthesis. This approach provides a robust framework for the development of safer and more efficacious FGF19 analogs.

Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency

Third-generation HIV-1-derived lentiviral vectors are successfully used as therapeutic agents in various clinical applications. To further promote their use, we attempted to enhance vector infectivity by targeting the dimerization and packaging properties of the RNA transfer vector based on the premise that these two processes are tightly linked. We rationally designed mutant vectors to favor the dimeric conformation, potentially enhancing genome packaging. Initial assessments using standard assays generated outputs of variable reproducibility, sometimes with conflicting results. Therefore, we developed a novel competitive qRT-PCR assay in a co-transfection setting to measure the relative packaging efficiencies of wild-type and mutant transfer vectors. Here we report the effect of the dimerization-stabilizing mutations on infectious and physical titers of lentiviral vectors together with their packaging efficiency, measured using our novel assay. Enhancing dimerization did not automatically lead to better vector RNA packaging, suggesting that, for vector functionality, sufficient flexibility of the RNA to adopt different conformations is more important than the dimerization capacity. Our novel competitive qPCR assay enables a more stringent analysis of RNA packaging efficiency, allowing a much more precise understanding of the links between RNA structure, packaging, and infectious titers that will be invaluable for future vector development.

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.

Overexpression of Human Mutant PANK2 Proteins Affects Development and Motor Behavior of Zebrafish Embryos.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a genetic and early-onset neurodegenerative disorder characterized by iron accumulation in the basal ganglia. It is due to mutations in Pantothenate Kinase 2 (PANK2), an enzyme that catalyzes the phosphorylation of vitamin B5, first and essential step in coenzyme A (CoA) biosynthesis. Most likely, an unbalance of the neuronal levels of this important cofactor represents the initial trigger of the neurodegenerative process, yet a complete understanding of the connection between PANK2 malfunctioning and neuronal death is lacking. Most PKAN patients carry mutations in both alleles and a loss of function mechanism is proposed to explain the pathology. When PANK2 mutants were analyzed for stability, dimerization capacity, and enzymatic activity in vitro, many of them showed properties like the wild-type form. To further explore this aspect, we overexpressed the wild-type protein, two mutant forms with reduced kinase activity and two retaining the catalytic activity in zebrafish embryos and analyzed the morpho-functional consequences. While the wild-type protein had no effects, all mutant proteins generated phenotypes that partially resembled those observed in pank2 and coasy morphants and were rescued by CoA and vitamin B5 supplementation. The overexpression of PANK2 mutant forms appears to be associated with perturbation in CoA availability, irrespective of their catalytic activity.

The JmjN domain as a dimerization interface and a targeted inhibitor of KDM4 demethylase activity.

Histone methylation is regulated to shape the epigenome by modulating DNA compaction, thus playing central roles in fundamental chromatin-based processes including transcriptional regulation, DNA repair and cell proliferation. Histone methylation is erased by demethylases including the well-established KDM4 subfamily members, however, little is known about their dimerization capacity and its impact on their demethylase activity. Using the powerful bimolecular fluorescence complementation technique, we herein show the in situ formation of human KDM4A and KDM4C homodimers and heterodimers in nuclei of live transfectant cells and evaluate their H3K9me3 demethylation activity. Using size exclusion HPLC as well as Western blot analysis, we show that endogenous KDM4C undergoes dimerization under physiological conditions. Importantly, we identify the JmjN domain as the KDM4C dimerization interface and pin-point specific charged residues therein to be essential for this dimerization. We further demonstrate that KDM4A/C dimerization is absolutely required for their demethylase activity which was abolished by the expression of free JmjN peptides. In contrast, KDM4B does not dimerize and functions as a monomer, and hence was not affected by free JmjN expression. KDM4 proteins are overexpressed in numerous malignancies and their pharmacological inhibition or depletion in cancer cells was shown to impair tumor cell proliferation, invasion and metastasis. Thus, the KDM4 dimer-interactome emerging from the present study bears potential implications for cancer therapeutics via selective inhibition of KDM4A/C demethylase activity using JmjN-based peptidomimetics.

Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcεRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ∼2700 IgE-FcεRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ≥8), although measurable responses were achieved when only a few hundred FcεRI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

Microphthalmia-associated transcription factor mutations are associated with white-spotted coat color in swamp buffalo.

SummaryA candidate gene analysis of the microphthalmia‐associated transcription factor (MITF) gene was used in an attempt to identify the genetic basis for a white‐spotted coat color phenotype in the Asian swamp buffalo (Bubalus bubalis carabanensis). Ninety‐three buffaloes—32 solid, 38 spotted and 23 white individuals—were Sanger‐sequenced for all MITF exons as well as highly conserved intronic and flanking regions. MITF cDNA representing skin and iris tissue from six spotted, nine solid and one white buffaloes was also Sanger‐sequenced to confirm detected mutations. Two independent loss‐of‐function mutations, a premature stop codon (c.328C>T, p.Arg110*) and a donor splice‐site mutation (c.840+2T>A, p.Glu281_Leu282Ins8), both of which cause white‐spotted coat color in swamp buffaloes, were identified. The nonsense mutation leads to a premature stop codon in exon 3, and likely removal of the resulting mRNA via nonsense‐mediated decay pathway, whereas the donor splice‐site mutation leads to aberrant splicing of exon 8 that encodes part of a highly conserved region of MITF. The resulting insertion of eight amino acid residues is expected to perturb the leucine zipper part in the basic helix‐loop‐helix leucine zipper (bHLH‐Zip) domain and will most likely influence dimerization and DNA binding capacity. Electrophoretic mobility shift assay was performed using mutant and wild‐type MITF proteins and showed that the mutant MITF protein resulting from the splice‐site mutation decreased in vitro DNA binding capacity compared to wild‐type MITF. White‐spotted buffalo bulls are sacrificed in funeral ceremonies in Tana Toraja, Indonesia, because they are considered holy, and our results show that genetic variation causes a tie to the cultural use of these buffaloes.

S-nitrosylation of mouse galectin-2 prevents oxidative inactivation by hydrogen peroxide

Galectins are a group of animal lectins characterized by their specificity for β-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione, a physiologically relevant S-nitrosylating agent. In the present study, recombinant mouse (m)Gal-2 was S-nitrosylated using nitrosocysteine (CysNO), which had no effect on the sugar-binding specificity and dimerization capacity of the protein. On the other hand, mGal-2 oxidation by H2O2 resulted in the loss of sugar-binding ability, while S-nitrosylation prevented H2O2-inducted inactivation, presumably by protecting the Cys residue(s) in the protein. These results suggest that S-nitrosylation by nitric oxides protect Gal-2 from oxidative stress in the gastrointestinal tract.

Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

The Role of Arg445 and Asp498 in the Human Thyroid Hormone Transporter MCT8

Monocarboxylate transporter 8 (MCT8) facilitates cellular influx and efflux of the thyroid hormones (THs) T(4) and T(3). Mutations in MCT8 lead to severe psychomotor retardation. Here, we studied the importance of 2 highly conserved residues (Arg445 in transmembrane domain 8 and Asp498 in transmembrane domain 10) for substrate recognition and helix interactions. We introduced single and double mutations (R445A, R445C, R445D, R445K, D498A, D498E, D498N, D498R, R445A+D498A, R445D+D498R, and R445K+D498E) in human MCT8 cDNA and studied the effects on MCT8-mediated TH uptake and metabolism in transfected cells. The impact of these mutations on MCT8 protein expression, dimerization capacity, and subcellular localization was studied by Western blotting and confocal microscopy. We found that mutations in Arg445 or Asp498 that alter the local charge resulted in a near-complete loss of TH uptake capacity, whereas the expression, stability, and subcellular localization of these mutant proteins was similar to those for wild-type MCT8. Given the impaired TH uptake, TH efflux could not be adequately studied. The importance of opposite charges at Arg445 and Asp498 was studied by exchanging these residues (R445D+D498R). In particular, T(4) uptake was less severely reduced by the exchange mutation than by the single mutations. Mutations of Arg445 and Asp498 to equally charged residues (R445K and/or D498E) resulted in TH uptake levels similar to wild-type MCT8. The presence of 2 oppositely charged residues at positions Arg445 and Asp498 that are predicted in close structural proximity is crucial for efficient TH uptake, which may indicate the presence of an, at least transient, charge pair between these residues.

Common γ Chain (γc) Cytokine Hypersensitivity Leads To T-Cell Homeostatic Deregulation Independent Of STAT3 Mutation In Large Granular Lymphocyte Leukemia

BackgroundT-cell receptor signaling along with intermittent homeostatic proliferation governed by STAT3/5-associated common γ chain (γc) cytokines IL-6, IL-2, IL-7 and IL-15 are the principal source of new T-cells after thymic involution. Persistent chronic infections and cancer lead to T-cell exhaustion characterized by a number of “exhaustion”-associated phenotypic changes and activation of a specific gene expression profile associated with negative regulation of cytokine signaling response over time. Lymphoproliferation, as seen in large granular lymphocyte leukemia, results from excessive proliferation in response to one or more of these cytokines leading to in vivo expansion. We showed previously using electrophoretic mobility shift assays (EMSA) that most LGL leukemia patients display constitutively active STAT3 DNA binding activity. Recently, an international group discovered that 30-40 % of patients have somatic mutations in STAT3. This provides new molecular insight into the basis of LGL leukemia, but does not resolve some key questions about the biology. First, leukemic LGL cells are not fully immortalized by the activation or mutation of STAT3 suggesting that their survival and expansion may be microenvironmentally regulated. Second, it is unclear if the STAT3 mutation is acquired within the clonal cell population that invariably possesses an “exhaustion-associated” CD57+/CD8+/CD28-/CD62L-/CD27- memory phenotype. Here, we determined the role of cytokine signaling in LGL leukemia. MethodsTo gain insight into the disease, we examined primary human T-cells from healthy controls (N=28) and LGL leukemia patients (N=28). In the LGL leukemia cohort, 14 patients harbored one of the recurring STAT3 mutations within the SH2 domain (Y640F, D661Y, D661H, D661V). The remaining LGL leukemia patients had no evidence of genomic abnormalities. Using these primary cells, we examined proliferation potential and pSTAT3/5 activation by flow cytometry (phosFlow) using methods published previously by our group. Signaling was determined after culture with 0.01, 0.1, and 1 ng/ml of IL-6 to activate STAT3, and similar doses of IL-2, IL-7 and IL-15 to activate STAT5. ResultsFirst, LGL leukemia cells showed hyperproliferative responses to the lowest dose of IL-15. Comparing CD57+ and CD57- T-cells in healthy individuals, CD57+ T-cells were hyporesponsive to all cytokines with regard to proliferation and phosphorylation in line with their exhausted phenotype. Next, we examined cytokine-mediated phosphorylation of STAT3 and STAT5 in LGL cases. STAT3 was normally phosphorylated in response to IL-6 in LGL leukemia T-cells. In contrast, hypersensitive STAT5 phosphorylation in response to the lowest dose of IL-6 (p=0.05), IL-2 (p=0.04), IL-7 (p=0.01), and IL-15 (p=0.02) occurred within CD57- T-cells. This occurred equally in both STAT3 mutant and non-mutant patient groups. The traditional CD57+ leukemic T-cell population was also hypersensitive to 100 fold-lower doses of IL-7 and IL-15 compared to healthy donors and again this difference was independent of the STAT3 mutation status. ConclusionLGL leukemia T-cells bypass protective mechanisms associated with T-cell exhaustion and display proliferation potential and hypersensitivity to STAT3/5-associated common γ chain (γc) cytokines. Similar differences in signaling were observed in both STAT3 mutant and non-mutant cases suggesting that it is a unifying abnormality. Although STAT3 was not hyperphosphorylated, it is possible that STAT3 is activated by enhanced dimerization capacity (STAT3-SH2 domain mutation) or through lack of negative regulators such as a deficiency in SOCS proteins. Our data, however, introduces a novel concept in this disease and suggests that the driving event lies within the CD57- population, where hypersensensitive STAT5 phosphorylation was evident in response to all cytokines. These results suggest that the driver mutation or event possibly resides within the common lymphoid progenitor population and that the presence of clonally-expanded CD57+ cells may result from incomplete exhaustion reprogramming following excessive cytokine-mediated proliferation within the lymphoid population. Disclosures:No relevant conflicts of interest to declare.