Expression of biologically active human interferon alpha 2 in Aloe vera.

Methods necessary for the successful transformation and regeneration of Aloe vera were developed and used to express the human protein, interferon alpha 2 (IFNα2). IFNα2 is a secreted cytokine that plays a vital role in regulating the cellular response to viral infection. Transgenic plants were regenerated from callus cultures initiated from zygotic embryos.

Studies of the ubiquitin proteasome system.

A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first-identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation.

Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics.

The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer.

RING-dependent tumor suppression and G2/M arrest induced by the TRC8 hereditary kidney cancer gene.

TRC8/RNF139 and von Hippel-Lindau (VHL) both encode E3 ubiquitin (Ub) ligases mutated in clear-cell renal carcinomas (ccRCC). VHL, inactivated in nearly 70% of ccRCCs, is a tumor suppressor encoding the targeting subunit for a Ub ligase complex that downregulates hypoxia-inducible factor-alpha. TRC8/RNF139 is a putative tumor suppressor containing a sterol-sensing domain and a RING-H2 motif essential for Ub ligase activity.

E6AP mediates regulated proteasomal degradation of the nuclear receptor coactivator amplified in breast cancer 1 in immortalized cells.

The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation.

Expression and evaluation of RING finger proteins.

RING finger proteins represent the largest class of potential ubiquitin ligases. This chapter describes methods used to express and assess the activity of proteins containing RING fingers based on our experience with a number of different family members. In addition to general protocols for assessing activity, specific protocols are provided for evaluating the ubiquitylation of p53 by the RING finger E3 Hdm2/Mdm2.

Expression, purification, and properties of the Ubc4/5 family of E2 enzymes.

Ubiquitin-conjugating enzymes (E2s) play a central role in ubiquitylation. They function to bridge the first, nonspecific step of ubiquitin activation by E1 with the transfer of activated ubiquitin to substrates by substrate-specific E3s. While sharing a common core UBC domain, members of this family exhibit significant specificity in their physical and functional interactions with E3s.

Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex.

The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation.

Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity.

SNURF/RNF4 has been implicated in transcriptional regulation and growth inhibition in a RING finger-dependent fashion. In this work, we show that SNURF mediates its own ubiquitination in vitro in a ubiquitin-conjugating enzyme (E2)-selective manner: SNURF acts as an E3 ligase with UbcH5A and B, HHR6B (RAD6B), E2-25K, MmUbc7 and UbcH13, but not with UbcH3, UbcM4, MmUbc6 or E2-20K. In contrast, the well-characterized RING E3, AO7, functions only with members of the UbcH5 family.

Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta.

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase.

RING finger ubiquitin protein ligases: implications for tumorigenesis, metastasis and for molecular targets in cancer.

Covalent modification of proteins with ubiquitin regulates almost all aspects of eukaryotic cellular function. Ubiquitin protein ligases (E3s) play central regulatory roles in that they provide substrate specificity to this process and therefore, represent attractive molecular targets for disease therapy. We summarize recent advances in our understanding of RING finger and RING finger-related E3s with emphasis on BRCA1 and the tumor autocrine motility factor receptor (gp78), as well as discuss the potential for components of the ubiquitin pathway for proteasomal degradation as molecular targets.

RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.

A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters retinoic acid receptor function in human keratinocytes.

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on all-trans-retinoic acid (trans-RA) binding to retinoic acid receptors (RARs) in cultured human keratinocytes (SCC-12F) was investigated. TCDD and trans-RA elicited opposing actions on the production of biologically active TGF-beta. TCDD exposure caused concentration- and time-dependent decreases in trans-RA binding to SCC-12F RARs.