Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.

The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function

A Regulatory Circuit Mediating Convergence between Nurr1 Transcriptional Regulation and Wnt Signaling▿

The orphan nuclear receptor Nurr1 is essential for the development and maintenance of midbrain dopaminergic neurons, the cells that degenerate during Parkinson's disease, by promoting the transcription of genes involved in dopaminergic neurotransmission. Since Nurr1 lacks a classical ligand-binding pocket, it is not clear which factors regulate its activity and how these factors are affected during disease pathogenesis. Since Wnt signaling via β-catenin promotes the differentiation of Nurr1+ dopaminergic precursors in vitro, we tested for functional interactions between these systems. We found that β-catenin and Nurr1 functionally interact at multiple levels. In the absence of β-catenin, Nurr1 is associated with Lef-1 in corepressor complexes. β-Catenin binds Nurr1 and disrupts these corepressor complexes, leading to coactivator recruitment and induction of Wnt- and Nurr1-responsive genes. We then identified KCNIP4/calsenilin-like protein as being responsive to concurrent activation by Nurr1 and β-catenin. Since KCNIP4 interacts with presenilins, the Alzheimer's disease-associated proteins that promote β-catenin degradation, we tested the possibility that KCNIP4 induction regulates β-catenin signaling. KCNIP4 induction limited β-catenin activity in a presenilin-dependent manner, thereby serving as a negative feedback loop; furthermore, Nurr1 inhibition of β-catenin activity was absent in PS1−/− cells or in the presence of small interfering RNAs specific to KCNIP4. These data describe regulatory convergence between Nurr1 and β-catenin, providing a mechanism by which Nurr1 could be regulated by Wnt signaling

Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC50, 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5α-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands