Shitao Zou

Abstract Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels 1–3 . A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown 4–7 . Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate 8–10 , enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14–ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation 11–13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies.

B7‑H3, a newly identified co‑stimulatory molecule, has been reported to be highly expressed in a number of types of cancer and is associated with a poor prognosis. Transwell experiments and a wound-healing assay were used to detect the role of over‑expressed B7‑H3 on cell migration and invasion in colorectal cancer (CRC) cells. The expression level of matrix metallopeptidase 9 (MMP‑9) was further investigated by zymography experiments and western blot analysis, and involvement of the Janus kinase 2 (Jak2) signal transducer and activator of transcription 3 (STAT3) signaling pathway was determined using AG490, a Jak2 selective inhibitor. Data showed that overexpression of B7‑H3 promoted cell migration and invasion in CRC. Further investigation certified that enhanced expression of B7‑H3 elevated MMP‑9 through upregulation of the Jak2‑Stat3 signaling pathway. Due to its pro‑migratory and pro‑invasive function, B7‑H3 may serve as a therapeutic target in the treatment of CRC.

BTG1, which belongs to the BTG/Tob family, regulates cell cycle progression in a variety of cell types and appears to play roles in inhibiting proliferation, promoting apoptosis and stimulating cellular differentiation in multiple cell types. However, it remains unclear whether BTG1 is a breast cancer suppressor gene, and the role of BTG1 in breast cancer cell growth has not yet been determined. In the present study, we observed that BTG1 was weakly expressed in human breast tumors and in breast cancer cells (MCF-7 and MDA-MB-231). In addition, we investigated the potential effects of BTG1 on breast cancer cell proliferation, cell cycle distribution and apoptosis after stable transfection with the BTG1 expression vector. We found that overexpression of BTG1 inhibited cell proliferation, induced G0/G1 cell cycle arrest and promoted apoptosis. Further investigation indicated that overexpression of BTG1 was involved in the inhibition of the expression of cell cycle-related proteins, cyclin B1 and cyclin D1, and pro-apoptotic factors, Bax and caspase-3, and was also involved in the promotion of anti-apoptotic factor Bcl-2. In vivo, animal experiments showed that tumors overexpressing BTG1 displayed a slower growth rate than the control xenografts. TUNEL end staining assay revealed that BTG1 induced tumor necrosis and apoptosis. Taken together, our data revealed that, in breast cancer cells, BTG1 inhibits cell growth through induction of cell cycle arrest and apoptosis. These results indicate that BTG1 may be used as a novel therapeutic target for human breast cancer treatment.

The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.