Anna Tretiakova

Regulation of myelin basic protein gene transcription by Sp1 and Purα: Evidence for association of Sp1 and Purα in brain

Direct interaction between transcription factors may provide a mechanism for the regulatory function of these proteins on transcription of the responsive genes. These interactions may be facilitated if the target DNA sequences for the participant regulatory proteins are overlapped or positioned in close proximity to each other within the promoter of the responsive genes. In earlier studies, we identified a cellular protein, named Purα, which upon binding to the MB1 regulatory DNA sequence of the myelin basic protein (MBP) gene, stimulates its transcription in central nervous system (CNS) cells. Here, we provide evidence for binding of the ubiquitous DNA binding transcription factor, Sp1, to the MB1 DNA motif at the region that partially overlaps with the Purα binding site. We demonstrate that binding of Purα to its target sequence is enhanced by inclusion of Sp1 in the binding reaction. Under this condition, binding of Sp1 to the MB1 regulatory sequence remained fairly unchanged, and no evidence for the formation of Purα:MB1:Sp1 was observed. This observation suggests that transient interaction of Purα and Sp1 may result in stable association of Purα and the MB1 element. In support of this notion, results from immunoprecipitation/Western blot studies have established association of Purα and Sp1 in nuclear extracts from mouse brain. Of interest, Purα appears to bind to the phosphorylated form of Sp1 which is developmentally regulated and that coincides with the periods when MBP gene expression is at its maximum level. Results from cotransfection studies revealed that ectopic expression of Purα and Sp1 synergistically stimulates MBP promoter activity in CNS cells. The importance of these findings in stage‐specific expression of MBP during brain development is discussed. J. Cell. Physiol. 181:160–168, 1999.

5-HT1B receptors play a prominent role in the proliferation of T-lymphocytes

Serotonin plays a role in T cell activation, but there is no clear consensus of which of the 14 serotonergic receptors control this activations pathway. We have used a broad range of serotonergic receptor antagonists to define the functional involvement of these receptors governing the proliferation of primary T cells as well as in T cell lines. Our data shows that antagonism of the 5-HT(1B) receptor inhibits the proliferation of both human and murine primary helper T cells and of human helper T cell lines. As a whole, our data suggest that other serotonergic receptors may contribute to the proliferative signals, but the 5-HT(1B) receptor plays the most dominant role.

Association of Purα and E2F-1 suppresses transcriptional activity of E2F-1

Protein-protein interaction can play an important role in the control of several biological events including gene transcription, replication and cell proliferation. E2F-1 is a DNA-binding transcription factor which, upon interaction with its target DNA sequence, induces expression of several S phase specific genes allowing progression of the cell cycle. Evidently, the activity of this protein is modulated by its cellular partner, pRb, which in the hypophosphorylated form, binds to E2F-1 and inactivates its transcriptional ability. In this study, we have demonstrated that expression of a sequence-specific single-stranded DNA binding protein, Purα, in cells decreases the ability of E2F-1 to exert its transcriptional activity upon the responsive promoter derived from DHFR. Results from band shift experiments revealed that while Purα does not recognize the double-stranded DNA fragment containing the E2F-1 binding site, it has the ability to inhibit E2F-1 interaction with its target DNA sequence. Results from GST pull-down assays and the combined immunoprecipitation/Western blot analysis of nuclear extracts revealed a direct association of E2F-1 with Purα in the absence of the DNA molecule containing the E2F-1 binding site. The association of Purα with E2F-1 may increase the stability of E2F-1, as a higher level of E2F-1 was detected in cells co-expressing Purα and E2F-1. The importance of these observations with respect to the role of Purα in the control of cell cycle progression is discussed.

Human neurotropic JC virus early protein deregulates glial cell cycle pathway and impairs cell differentiation.

Progressive multifocal leukoencephalopathy (PML), a human demyelinating disease of the central nervous system (CNS), is induced upon replication of the human neurotropic virus, JCV, in glial cells. Similar to other polyomaviruses, replication of JCV is initiated and orchestrated by the viral early protein, T‐antigen, and results in the cytolytic destruction of oligodendrocytes, the subset of glial cells responsible for myelin production, and the appearance of bizarre astrocytic glial cells in affected individuals. Earlier results from studies in transgenic animals have suggested that in the absence of viral replication, expression of JCV T‐antigen induces pathology consistent with hypomyelination of the brain. These observations suggest that JCV T‐antigen has the ability to deregulate oligodendrocyte and perhaps astrocyte function in the CNS. Here we demonstrate that expression of JCV T‐antigen in the bipotential glial cell line, CG‐4, severely affects the ability of these cells to differentiate toward oligodendrocyte and astrocyte lineages as evidenced by their distinct morphological changes. Examination of the activity of cell cycle regulatory proteins including cyclins and their associated kinases reveals that in the absence of T‐antigen, differentiation of CG‐4 cells toward astrocytes and oligodendrocytes is accompanied by a decline in cyclin E, cdk2, cyclin A, and cyclin B activity. In contrast, cdc2 activity increased upon CG‐4 differentiation. In T‐antigen‐producing cells, distinct variations in the activity of several cyclins was observed. For example, while the activity of cdk2 and cyclin E was enhanced in T‐antigen expressing astrocytes compared to their levels in control cells, the activity of cdc2 was decreased in this cell type. In oligodendrocytes, expression of T‐antigen decreased the activity of several cyclins and cdks including cyclin E and cdc2. On the other hand, the level of expression and activity of cyclin A was increased. Thus, it is evident that JCV T‐antigen deregulates several important cell cycle regulators during CG‐4 differentiation, and these alterations may contribute to the process of cell growth and differentiation in glial cells. The importance of our findings with regard to the neuropathogenesis of PML is discussed. J. Neurosci. Res. 55:588–599, 1999.u2003

Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low kcat despite a tight Km. Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X‐ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site‐directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had kcat/Km less than 10% of the wild‐type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3‐ to 12‐fold increase of Km for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9‐ to 200‐fold increase of Ki for cGMP in comparison to the wild‐type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.

Compatibility of detergents with the microbatch-under-oil crystallization method.

Detergents are required to solubilize integral membrane proteins and are common components of the solutions used to crystallize these molecules. It has been unclear whether these detergents are completely compatible with the oils used in the microbatch-under-oil crystallization technique, because they might conceivably be lost from solution by partitioning into the oil phase. The partitioning of the detergents n-octyl-beta-d-glucopyranoside and Fos-Choline-12 into two different oils used for microbatch crystallization experiments has been examined. It was found that vigorous mixing and prolonged incubation of the aqueous detergent solutions with the oils leads to small losses of detergent (approximately 5% of the total detergent mass); however, gentle mixing that is more typical of the mixing encountered in a crystallization experiments leads to negligible loss of detergent.

Rational design of cytotoxic T-cell inhibitors.

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 α-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analogs activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.

Structure and function of HIV-1 and SIV Tat proteins based on carboxy-terminal truncations, chimeric Tat constructs, and NMR modeling

To further define the structure and function of the domains in HIV-1 and SIV Tat proteins, chimeric Tat cDNA expression constructs were generated with crossover points at the carboxy-terminal end of the cysteine rich domain. The chimera containing the amino-terminal region of SIV and carboxy-terminal region of HIV exhibited activity similar to HIV-1 Tat and SIV Tat on both the HIV-1 and SIV LTRs. In contrast, the reciprocal chimera functioned poorly. As determined by the activity of carboxy-terminal truncation mutants, the region immediately downstream of the basic domain is critical for efficient transactivation by HIV-1 Tat, but not SIV Tat protein. In this report, we present a model for Tat domains based on NMR data and the known functional properties of Tat protein. According to our modeling two sites for protein : protein interactions are present in HIV-1 and SIV Tat proteins. Site I, which is presumably involved in cyclin T binding, is similar in both HIV-1 and SIV Tat proteins as well as in Tat chimeras. Site II, however appears structurally different in HIV-1 and SIV Tat models, although in both cases is comprised of amino and carboxy-terminal residues. Differences in Site II may thus account for the differential activities of HIV-1 and SIV Tat carboxy-terminal truncations. Site II in the poorly active chimera differs significantly from that found in HIV-1 and SIV Tat proteins. The two site structural model presented here may have important implications for the role of Tat in HIV pathogenesis and may provide insights for the design of Tat vaccines and targeted therapeutics.

A Rational Design Approach for Developing Immunomodulators Based on CD4 and CD8

The pharmaceutical industry has classically relied on screening methods for its discovery and development of new drugs. Moreover, the recent use of high throughput screening has radically changed the industrys ability to expand drug searches. These approaches have proven to be highly successful and account for> 90% of aU ofthe prescription drug market. From a Company perspective, the more compounds screened, the greater the likelihood that a new drug can be added to the ~pipeline. Although random screens are time-consuming and labour-intensive, these methods have a proven success record and are unlikely to change in the near future. Today~ the compound libraries are, generally, the products of shotgun combinatorial chemistry or are collections of natural products. The screens, by necessity, are designed for ease-of-use. Consequently, the initial system must have a simplified read-out for screening potential drug hits ~ Often, this means that the assay read-out has been reduced to monitoring the formation or disassociation of a protein complex or for monitoring the expression of an engineered marker such as luciferase, to screen for