A transcriptional activating region with two contrasting modes of protein interaction

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein–protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

On the induced electric field gradients in the human body for magnetic stimulation by gradient coils in MRI

Prior theoretical studies indicate that the negative spatial derivative of the electric field induced by magnetic stimulation may he one of the main factors contributing to depolarization of the nerve fiber. This paper studies this parameter for peripheral nerve stimulation (PNS) induced by time.-varying gradient fields during MRI scans. The numerical calculations are based on an efficient, quasi-static, finite-difference scheme and an anatomically realistic human, full-body model. Whole-body cylindrical and planar gradient sets in MRI systems and various input signals have been explored. The spatial distributions of the induced electric field and their gradients are calculated and attempts are made to correlate these areas with reported experimental stimulation data. The induced electrical field pattern is similar for both the planar coils and cylindrical coils. This study provides some insight into the spatial characteristics of the induced field gradients for PNS in MRI, which may be used to further evaluate the sites where magnetic stimulation is likely to occur and to optimize gradient coil design.

Calculation of electric fields induced by body and head motion in high-field MRI

In modern magnetic resonance imaging (MRI), patients are exposed to strong, nonuniform static magnetic fields outside the central imaging region, in which the movement of the body may be able to induce electric currents in tissues which could be possibly harmful. This paper presents theoretical investigations into the spatial distribution of induced electric fields and currents in the patient when moving into the MRI scanner and also for head motion at various positions in the magnet. The numerical calculations are based on an efficient, quasi-static, finite-difference scheme and an anatomically realistic, full-body, male model. 3D field profiles from an actively shielded 4T magnet system are used and the body model projected through the field profile with a range of velocities. The simulation shows that it possible to induce electric fields/currents near the level of physiological significance under some circumstances and provides insight into the spatial characteristics of the induced fields. The results are extrapolated to very high field strengths and tabulated data shows the expected induced currents and fields with both movement velocity and field strength. (C) 2003 Elsevier Science (USA). All rights reserved.

Peroxisome proliferator-activated receptor structures: ligand specificity, molecular switch and interactions with regulators.

Peroxisome proliferator-activated receptors (PPARs) compose a family of nuclear receptors that mediate the effects of lipidic ligands at the transcriptional level. In this review, we highlight advances in the understanding of the PPAR ligand binding domain (LBD) structure at the atomic level. The overall structure of PPARs LBD is described, and important protein ligand interactions are presented. Structure-activity relationships between isotypes structures and ligand specificity are addressed. It is shown that the numerous experimental three-dimensional structures available, together with in silico simulations, help understanding the role played by the activating function-2 (AF-2) in PPARs activation and its underlying molecular mechanism. The relation between the PPARs constitutive activity and the intrinsic stability of the active conformation is discussed. Finally, the interactions of PPARs LBD with co-activators or co-repressors, as well as with the retinoid X receptor (RXR) are described and considered in relation to PPARs activation.

Chromatin components as part of a putative transcriptional repressing complex

The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is a Drosophila protein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.

Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements.

The evolutionarily conserved Krüppel-associated box (KRAB) is present in the N-terminal regions of more than one-third of all Krüppel-class zinc finger proteins. Recent experiments have demonstrated that the KRAB-A domain tethered to a promoter DNA by connecting to heterologous DNA-binding protein domain or targeted to a promoter-proximal RNA sequence acts as a transcriptional silencing of RNA polymerase II promoters. Here we show that expression of KRAB domain suppresses in vivo the activating function of various defined activating transcription factors, and we demonstrate that the KRAB domain specifically silences the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription initiation is directed by a pyrimidine-rich initiator element, however, are relatively unaffected. We also report in vitro transcription experiments indicating that the KRAB domain is able to repress both activated and basal promoter activity. Thus, the KRAB domain appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Expression of an activating mutation in the gene encoding the K(ATP) channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K(+) (K(ATP)) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K(ATP) channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K(ATP) channels, closed K(ATP) channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of K(ATP) channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Increased number and function of natural killer cells in human immunodeficiency virus 1-positive subjects co-infected with herpes simplex virus 2

P>Natural killer (NK) cells bridge the interface between innate and adaptive immunity and are implicated in the control of herpes simplex virus 2 (HSV-2) infection. In subjects infected with human immunodeficiency virus 1 (HIV-1), the critical impact of the innate immune response on disease progression has recently come into focus. Higher numbers of NK cells are associated with lower HIV-1 plasma viraemia. Individuals with the compound genotype of killer cell immunoglobulin-like receptor (KIR) 3DS1 and human leucocyte antigen (HLA)-Bw4-80I, or who have alleles of KIR3DL1 that encode proteins highly expressed on the NK cell surface, have a significant delay in disease progression. We studied the effect of HSV-2 co-infection in HIV-1-infected subjects, and show that HSV-2 co-infection results in a pan-lymphocytosis, with elevated absolute numbers of CD4+ and CD8+ T cells, and NK cells. The NK cells in HSV-2 co-infected subjects functioned more efficiently, with an increase in degranulation after in vitro stimulation. The number of NK cells expressing the activating receptors NKp30 and NKp46, and expressing KIR3DL1 or KIR3DS1, was inversely correlated with HIV-1 plasma viral load in subjects mono-infected with HIV-1, but not in subjects co-infected with HSV-2. This suggests that HSV-2 infection mediates changes within the NK cell population that may affect immunity in HIV-1 infection.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells.

NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon gamma (IFNgamma) production, suggesting sustained signaling. The functional changes are associated with a reduced presence of the relevant signal transducing adaptors DNAX-activating protein of 10 kDa (DAP-10) and killer cell activating receptor-associated protein/DNAX-activating protein of 12 kDa (KARAP/DAP-12). That is likely the consequence of constitutive NKG2D engagement and signaling, since NKG2D function and adaptor expression is restored to normal when the stimulating tumor cells are removed. Thus, the chronic exposure to tumor cells expressing NKG2D ligand alters NKG2D signaling and may facilitate the evasion of tumor cells from NK cell reactions.

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.