The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Å. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.

This report shows for the first time the efficient uniform isotope labeling of a recombinant protein expressed using Baculovirus-infected insect cells. The recent availability of suitable media for (15)N- and (13)C/(15)N-labeling in insect cells, the high expression of Abl kinase in these labeling media and a suitable labeling protocol made it possible to obtain a (1)H-(15)N-HSQC spectrum for the catalytic domain of Abl kinase of good quality and with label incorporation rates > 90%. The presented isotope labeling method should be applicable also to further proteins where successful expression is restricted to the Baculovirus expression system.

The interleukin-1 beta-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 beta precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.

The majority of menopausal women suffer from climacteric symptoms. The purpose of this study was to assess the effects of acupuncture on the quality of life and reproductive hormones secretion in menopausal women. Eleven (11) menopausal women with climacteric symptoms entered this prospective study. The Menopause Specific Quality of life Questionnaire was filled out by the patients before the first acupuncture session, after the last one (5 weeks later), and 3 months after the last acupuncture session. Reproductive hormones including follicular-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, and prolactin were measured before and after treatment. Acupuncture significantly improved menopausal vasomotor symptoms (p = 0.001 and p = 0.003 for the end of treatment and 3 months later, respectively) and physical symptoms (p = 0.014 at the end of treatment and p = 0.046 3 months later). It did not change psychosocial or sexual symptoms, nor did it change the measured reproductive hormones. In conclusion, acupuncture is shown to be effective in relieving vasomotor and physical disturbances of menopausal women with effects lasting at least up to 3 months after termination of the treatment. Acupuncture may be a useful treatment alternative for women who are unable or do not want to receive hormone replacement therapy. A prospective study with larger sample sizes will be needed to define the role of acupuncture in the management of menopausal symptoms.

Signal transducers and activators of transcription (STAT) 1 and STAT3 are activated by overlapping but distinct sets of cytokines. STATs are recruited to the different cytokine receptors through their Src homology (SH) 2 domains that make highly specific interactions with phosphotyrosine-docking sites on the receptors. We used a degenerate phosphopeptide library synthesized on 35-m TentaGel beads and fluorescenceactivated bead sorting to determine the sequence specificity of the peptide-binding sites of the SH2 domains of STAT1 and STAT3. The large bead library allowed not only peptide sequencing of pools of beads but also of single beads. The method was validated through surface plasmon resonance measurements of the affinities of different peptides to the STAT SH2 domains. Furthermore, when selected peptides were attached to a truncated erythropoietin receptor and stably expressed in DA3 cells, activation of STAT1 or STAT3 could be achieved by stimulation with erythropoietin. The combined analysis of pool sequencing, the individual peptide sequences, and plasmon resonance measurements allowed the definition of SH2 domain binding motifs. STAT1 preferentially binds peptides with the motif phosphotyrosine-(aspartic acid/glutamic acid)-(proline/ arginine)-(arginine/proline/glutamine), whereby a negatively charged amino acid at ؉1 excludes a proline at ؉2 and vice versa. STAT3 preferentially binds peptides with the motif phosphotyrosine-(basic or hydrophobic)-(proline or basic)-glutamine. For both STAT1 and STAT3, specific high affinity phosphopeptides were identified that can be used for the design of inhibitory molecules.The signal transducers and activators of transcription (STATs) 1 constitute a family of latent cytoplasmic transcription factors that are activated by a large number of cytokines, growth factors, and hormones. The binding of these extracellular signaling polypeptides to specific cell surface receptors typically results in receptor homo-or heterodimerization and consecutive activation of receptor-associated protein tyrosine kinases of the Jak family. Activated Jak kinases phosphorylate tyrosine residues in the intracellular domains of the receptors (1). STATs then bind with their SH2 domains to these receptordocking sites. The Jak kinases phosphorylate the STATs on a single tyrosine located carboxyl-terminal to the SH2 domain (2). The tyrosine phosphorylation of STATs is the decisive activation event, resulting in STAT dimer formation through mutual SH2 domain-phosphotyrosine interactions. STAT dimers translocate into the nucleus, bind to response elements in gene promoters, and enhance the transcription of these target genes (3-5). Seven mammalian STAT genes have been identified in three chromosomal clusters (6). The different STAT proteins are activated by distinct cytokines and growth factors, and each STAT protein activates a distinct set of target genes (5, 7). The specific coupling of the different STAT family members to cytokine receptors is crucial for the generation of diverse intracellu...