Chaperone-mediated 26S Proteasome Remodeling Facilitates Free K63 Ubiquitin Chain Production and Aggresome Clearance

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.     

Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL–mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies. R.M. Reddy and W.-S. Yeow contributed equally to this work. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH.     

N-(2-Amino-phenyl)-4-(heteroarylmethyl)-benzamides as new histone deacetylase inhibitors

A variety of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benzamides were designed and synthesized. These compounds were shown to inhibit recombinant human HDAC1 with IC₅₀ values in the sub-micromolar range. In human cancer cells growing in culture these compounds induced hyperacetylation of histones, induced the expression of the tumor suppressor protein p21^WAF1/Cip1, and inhibited cellular proliferation. Certain compounds of this class also showed in vivo activity in various human tumor xenograft models in mice.     

Is Sirt1 a miracle bullet for longevity?

The Sir2 (silent information regulator 2) family of nicotinamide adenine dinucleotide-dependent deacetylases has been implicated in the regulation of aging and longevity across a wide variety of organisms. Although controversial, Sir2 proteins have also been implicated as key mediators for the beneficial effects of caloric restriction (CR) on aging and longevity. In this issue, Bordone et al . report that transgenic mice in which the mammalian Sir2 ortholog Sirt1 is overexpressed mimic the physiological changes in response to CR. These findings have important implications for the development of CR mimetics and perhaps also for lifespan extension.     

Induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through HDAC-independent and IP3-mediated PRKAA activation

Autophagy is closely related to tumor cell sensitivity to anticancer drugs. The HDAC (histone deacetylase) inhibitor valproic acid (VPA) interacted synergistically with chemotherapeutic agents to trigger lymphoma cell autophagy, which resulted from activation of AMPK (AMP-activated protein kinase) and inhibition of downstream MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling. In an HDAC-independent manner, VPA potentiated the effect of doxorubicin on lymphoma cell autophagy via reduction of cellular inositol 1,4,5 trisphosphate (IP3), blockade of calcium into mitochondria and modulation of PRKAA1/2-MTOR cascade. In murine xenograft models established with subcutaneous injection of lymphoma cells, dual treatment of VPA and doxorubicin initiated IP3-mediated calcium depletion and PRKAA1/2 activation, induced in situ autophagy and efficiently retarded tumor growth. Aberrant genes involving mitochondrial calcium transfer were frequently observed in primary tumors of lymphoma patients. Collectively, these findings suggested an HDAC-independent chemosensitizing activity of VPA and provided an insight into the clinical application of targeting autophagy in the treatment of lymphoma.