Biomedical subjects

Stefan N Constantinescu

Publications and source records attributed to Stefan N Constantinescu.

17 recordsLinked to original sources

PubMed2006

Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42.

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.

Amino Acid Sequence↗

PubMed2005

A JAK2 mutation in myeloproliferative disorders: pathogenesis and therapeutic and scientific prospects.

Myeloproliferative disorders include several pathologies sharing the common feature of being clonal hematopoietic stem cell diseases. The molecular basis of chronic myeloid leukemia was characterized many years ago with the discovery of the t(9;22) translocation and its product the BCR-ABL oncoprotein. The recent finding of a recurrent mutation in the Janus 2 tyrosine kinase gene is a major advance in our understanding of the pathogenesis of several other myeloproliferative disorders, including polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Although this work clearly identifies a frequent ( approximately 50%) subgroup of myeloproliferative disorders and explains most biological abnormalities described so far, it also raises the major question of how a single mutation can explain disease heterogeneity. Such a recurrent and unique mutation leading to a tyrosine kinase deregulation would make a suitable target for the development of specific therapies.

Gene Components↗

PubMed2005

An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor.

Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (delta5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, delta5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. Delta5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the delta5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces.

Amino Acid Motifs↗

PubMed2005

JAK1 and Tyk2 activation by the homologous polycythemia vera JAK2 V617F mutation: cross-talk with IGF1 receptor.

The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases.

Cell Line, Tumor↗

PubMed2005

Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells.

Mouse embryonic stem (ES) cells can be propagated in vitro while retaining their properties of pluripotency and self-renewal under the continuous presence of leukemia inhibitor factor (LIF). An essential role has been attributed to subsequent activation of the Stat3 transcription factor in mediating LIF self-renewal response. To date, however, downstream target genes of Stat3 in ES cells are still unknown. To isolate these genes, we performed a microarray-based kinetic comparison of LIF-stimulated (undifferentiated) ES cells versus ES cells induced to differentiate by shutting down Stat3 activity through either LIF deprivation or, more specifically, expression of a Stat3 dominant-negative mutant. In each case, we chose the earliest time at which ES cells lose their self-renewal properties, as illustrated by a decrease in the number of embryoid bodies and blast cell colony formation as well as germ layer marker expression. Comparison of the two independent approaches revealed similarly regulated genes that are likely to be involved in the Stat3 effects on ES cell self-renewal. For instance, upregulation of growth factors such as the transforming growth factor-beta relative Lefty1 or transcriptional regulators such as Id1 and Id2 and down-regulation of the groucho-like protein Aes1 (grg5) were found. Promoter analysis of the aes1 gene revealed three functional Stat3 consensus sites, as shown by luciferase assays. Furthermore, chromatin immunoprecipitation experiment demonstrated that Stat3 is recruited to the promoter of aes1 in ES cells. These data demonstrated that the aes1 gene is a direct transcriptional target of Stat3 in ES cells.

Animals↗

PubMed2005

Janus kinases affect thrombopoietin receptor cell surface localization and stability.

The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR.

Animals↗

PubMed2005

Helix packing and orientation in the transmembrane dimer of gp55-P of the spleen focus forming virus.

gp55-P is a dimeric membrane protein with a single transmembrane helix that is coded by the env gene of the polycythemic strain of the spleen focus forming virus. gp55-P activates the erythropoietin (Epo) receptor through specific transmembrane helix interactions, leading to Epo-independent growth of erythroid progenitors and eventually promoting erythroleukemia. We describe the use of magic angle spinning deuterium NMR to establish the structure of the transmembrane dimer of gp55-P in model membranes. Comparison of the deuterium lineshapes of leucines in the center (Leu(396-399)) and at the ends (Leu(385), Leu(407)) of the transmembrane sequence shows that gp55-P has a right-handed crossing angle with Leu(399) packed in the dimer interface. We discuss the implications of the structure of the gp55-P transmembrane dimer for activation of the Epo receptor.

Cell Membrane↗

PubMed2005

A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.

Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

Animals↗

PubMed2005

Differential STAT5 signaling by ligand-dependent and constitutively active cytokine receptors.

Many leukemia and cancer cells exhibit constitutive activation of STAT5, which was suggested to provide an anti-apoptotic advantage. Transformation of cytokine-dependent hematopoietic cells, such as Ba/F3 cells to autonomous growth and tumorigenicity equally results in selection for constitutive activation of STAT5. We compared STAT5 signaling between erythropoietin(Epo)-dependent cells and cells that were transformed by oncogenic activation of the erythropoietin receptor (EpoR) by coexpression of the gp55-P envelope protein of the spleen focus forming virus or by expression of the R129C constitutively active EpoR mutant. In transformed cells it was mainly STAT5B that was constitutively activated. In contrast, Epo stimulation activated both STAT5A and STAT5B. In transformed cells, chromatin immunoprecipitation (ChIP) showed STAT5 to be physically bound to promoters of STAT5 target genes, such as Bcl(XL), and to be able to promote transactivation of the Bcl(XL) promoter in a constitutive fashion. Sequencing of native sequences after ChIP with anti-STAT5 antibodies in Epo-dependent and -transformed cells indicated that in gp55-transformed cells, STAT5B bound in the chromatin not only to N3 high affinity, but also to low affinity N4 GAS sites. Transactivation for N3 GAS sites in luciferase reporters was specific to gp55 transformation. Because we also found preferential constitutive STAT5B activation after transformation of cells by a truncated form of the G-CSF-R that produces severe neutropenia (Kostmann syndrome) and favors leukemia in humans, we discuss the potential role of STAT5B in oncogenic transformation of hematopoietic cells.

Active Transport, Cell Nucleus↗

PubMed2005

Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function.

The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.

Amino Acid Motifs↗

PubMed2005

Role of tyrosine kinases and phosphatases in polycythemia vera.

Protein tyrosine kinases (PTKs) and phosphatases (PTPs) play a crucial role in normal cell development, and dysfunction of these enzymes has been implicated in human cancers. Polycythemia vera (PV) is a clonal hematologic disease characterized by hypersensitivity of hematopoietic progenitor cells to growth factors and cytokines. Recently, a unique and clonal mutation in the JAK homology 2 (JH2) domain of JAK2 that results in a valine to phenylalanine substitution at position 617 (V617F) was found in the majority of PV patients. This mutation leads to constitutive JAK2 activation and abnormal signaling and induces erythrocytosis in an animal model. The mutation is also found in a significant percentage of patients with idiopathic myelofibrosis (50%) and essential thrombocythemia (30%). Thus, it seems probable that this mutation associates with other molecular genetic events to cause different myeloproliferative disorders (MPDs). One of these secondary events is the transition to homozygosity of the mutated gene in 30% of the PV patients. Other events may include defects in PTPs, but these remain to be characterized. Recent studies represent a great step forward in the molecular pathogenesis in PV and the development of targeted new drugs to treat the disease.

Hematopoietic Stem Cells↗

PubMed2005

A unique activating mutation in JAK2 (V617F) is at the origin of polycythemia vera and allows a new classification of myeloproliferative diseases.

Myeloproliferative disorders (MPDs) are heterogeneous diseases that occur at the level of a multipotent hematopoietic stem cell. They are characterized by increased blood cell production related to cytokine hypersensitivity and virtually normal cell maturation. The molecular pathogenesis of the MPDs has been poorly understood, except for chronic myeloid leukemia (CML), where the Bcr-Abl fusion protein exhibits constitutive kinase activity. Since some rare MPDs are also related to a dysregulated kinase activity, a similar mechanism was thought to be likely responsible for the more frequent MPDs. We investigated the mechanisms of endogenous erythroid colony formation (EEC) by polycythemia vera (PV) erythroid progenitor cells and found that EEC formation was abolished by a pharmacological inhibitor of JAK2 as well as an siRNA against JAK2. JAK2 sequencing revealed a unique mutation in the JH2 domain leading to a V617F substitution in more than 80% of the PV samples. This mutation in the pseudokinase autoinhibitory domain results in constitutive kinase activity and induces cytokine hypersensitivity or independence of factor-dependent cell lines. Retroviral transduction of the mutant JAK2 into murine HSC leads to the development of an MPD with polycythemia. The same mutation was found in about 50% of patients with idiopathic myelofibrosis (IMF) and 30% of patients with essential thrombocythemia (ET). Using different approaches, four other teams have obtained similar results. The identification of the JAK2 mutation represents a major advance in our understanding of the molecular pathogenesis of MPDs that will likely permit a new classification of these diseases and the development of novel therapeutic approaches.

Amino Acid Substitution↗

PubMed2004

High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of STAT5 overexpression.

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 5' of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning.

Animals↗

PubMed2003

The erythropoietin receptor transmembrane domain mediates complex formation with viral anemic and polycythemic gp55 proteins.

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A-->P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region.

Amino Acid Sequence↗

PubMed2003

Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer.

Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.

Amino Acid Sequence↗

PubMed2003

Development of human protein reference database as an initial platform for approaching systems biology in humans.

Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.

BRCA1 Protein↗

PubMed2002

A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling.

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.

Adaptor Proteins, Signal Transducing↗