Biomedical subjects

B J Calnan

Publications and source records attributed to B J Calnan.

8 recordsLinked to original sources

PubMed1995

A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection.

The transcription factor Nur77, an orphan member of the nuclear hormone receptor superfamily, is highly expressed during T cell receptor-signaled apoptosis, suggesting a possible role for Nur77 in negative selection. We examined this by generating two sets of transgenic mice. In one set of mice, a dominant-negative Nur77 mutant is constitutively expressed and the other in which wild-type Nur77 protein is constitutively expressed in developing thymocytes. We report that inhibition of endogenous Nur77 by the dominant-negative mutant perturbed T cell development and inhibited antigen-induced negative selection in F5T cell receptor transgenic mice. Constitutive expression of wild-type Nur77 protein induced apoptosis in developing thymocytes, resulting in a decreased number of thymocytes and mature T cells. Together, these data support a role for Nur77 in the downstream signaling events in antigen-induced negative selection.

Animals↗

PubMed1995

Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis.

T-cell receptor (TCR)-mediated apoptosis in immature thymocytes and T-cell hybridomas is calcium dependent and can be inhibited by cyclosporin A (CsA). Induction of the orphan steroid receptor Nur77 (NGFI-B) is required for activation-induced apoptosis. Here, we examined the regulation of Nur77 expression, in response to apoptotic TCR signals, which consists of kinase C and calcium pathways. We show that the major control of Nur77 induction is mediated by the calcium signaling pathway. In contrast, protein kinase C signals induce only a low level of Nur77 activity. Nur77 promoter activity parallels its protein levels. CsA decreases both Nur77 protein levels and promoter activity, and the kinetics of CsA inhibition of apoptosis correlates with a decrease in Nur77 protein levels. TCR signals and kinase C signals result in a similar level of Nur77 protein phosphorylation but mediate differential transactivation activity of Nur77. In addition, Nur77 promoter deletion analysis revealed two RSRF (related to serum-responsive factor) binding sites, which can confer calcium and CsA sensitivity on a heterologous promoter. Taken together, our data suggest that the levels of transcriptional induction of Nur77 play an important role during activation-induced apoptosis and that calcium signals regulate a novel CsA-sensitive nuclear factor required for Nur77 transcription in T cells.

Animals↗

PubMed1992

Conformation of the TAR RNA-arginine complex by NMR spectroscopy.

The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.

Arginine↗

PubMed1992

Specific binding of a basic peptide from HIV-1 Rev.

Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents.

Amino Acid Sequence↗

PubMed1991

Arginine-mediated RNA recognition: the arginine fork.

Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R9) also bound specifically to TAR, and a mutant Tat protein that contained R9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K9) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine eta nitrogens and the epsilon nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures.

Amino Acid Sequence↗

PubMed1991

Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition.

Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.

Amino Acid Sequence↗

PubMed1990

HIV-1 Tat protein trans-activates transcription in vitro.

Tat protein of human immunodeficiency virus 1 is a potent trans-activator of viral gene expression. We show that purified Tat protein stimulates transcription from viral promoters greater than 10-fold in vitro. A Tat protein mutant that does not trans-activate in vivo did not stimulate transcription in vitro. Tat trans-activation required a functional TAR RNA sequence; trans-activation was competed by the addition of in vitro synthesized wild-type TAR RNA but not by mutant TAR RNAs. That Tat protein directly interacts with the TAR RNA during trans-activation in vitro was suggested by competition with Tat peptides. Preliminary evidence suggests the involvement of a cellular factor in recognition of TAR RNA during Tat trans-activation. Analysis of Tat trans-activation in vitro will provide new mechanistic insights into this process and allow a more detailed study of the relationship between Tat protein structure and function.

Amino Acid Sequence↗

PubMed1984

Characterization of endemic Providencia stuartii isolates from patients with urinary devices.

Providencia stuartii has emerged as a significant nosocomial urinary tract pathogen. An increase in the number of Providencia isolates from urine cultures prompted an investigation into the possibility of an outbreak due to this organism. A high proportion of patients studied had urinary devices. Four wards were screened at two time periods to ascertain the prevalence of Providencia stuartii in urine cultures. Biotype, serotype, antibiogram and plasmid content were determined for each Providencia isolate. Of 129 patients initially sampled 22.5% were found to harbor Providencia stuartii. Biotyping, serotyping and antibiograms indicated an epidemic strain was not present. Similar results were obtained when the wards were screened a second time, with 25.4% of urine cultures found to contain Providencia stuartii. By plasmid analysis the isolates could be grouped into one of ten profiles. A correlation could be made between urease activity and the presence of a large plasmid. No association however could be made between a particular plasmid profile and antibiogram. The data indicate that an epidemic strain of Providencia stuartii was not present. The source(s) of the endemic Providencia stuartii strains remain unknown.

Bacteriuria↗