Restoration of senescence in breast and ovarian cancer cells following the transfer of the YAC carrying SEN6A gene located at 6q16.3.

We previously located a senescence gene locus (SEN6A), at chromosome 6q14-21 by a functional strategy using chromosome transfer into immortal ovarian tumor cells. To further elucidate the SEN6A locus, intact chromosome 6 or 6q was transferred into rat ovarian tumor cells and a panel of immortal revertant clones of senescent cells was generated. The panel of independent colonies as well as mixed populations of revertant cells was analyzed for the presence or absence of chromosome 6 specific markers. These investigations led to the identification of a fine deletion of approximately 1cM at chromosomal interval 6q16.3. A contiguous stretch containing five yeast artificial chromosome (YAC) clones was constructed across the deleted region. The non-chimeric YAC clones were retrofitted and transferred into mouse A9 cells by spheroplast fusion to generate YAC/A9 hybrids. YAC DNA present in YAC/A9 hybrids was subsequently transferred by microcell fusion into immortal tumor cells, and the hybrid cells were characterized for their senescence phenotype. Using this functional strategy, the transfer of YAC clone 966b10 was shown to restore senescence in both rat and human ovarian and breast tumor cells. Our results demonstrate that the SEN6A gene is carried on a 1 Mb YAC, 966b10, which maps at 6q16.3.

Cytosolic aggregates perturb the degradation of nontranslocated secretory and membrane proteins.

A wide range of diseases are associated with the accumulation of cytosolic protein aggregates. The effects of these aggregates on various aspects of normal cellular protein homeostasis remain to be determined. Here we find that cytosolic aggregates, without necessarily disrupting proteasome function, can markedly delay the normally rapid degradation of nontranslocated secretory and membrane protein precursors. In the case of mammalian prion protein (PrP), the nontranslocated fraction is recruited into preexisting aggregates before its triage for degradation. This recruitment permits the growth and persistence of cytosolic PrP aggregates, explaining their apparent "self-conversion" seen in earlier studies of transient proteasome inhibition. For other proteins, the aggregate-mediated delay in precursor degradation led to aggregation and/or soluble residence in the cytosol, often causing aberrant cellular morphology. Remarkably, improving signal sequence efficiency mitigated these effects of aggregates. These observations identify a previously unappreciated consequence of cytosolic aggregates for nontranslocated secretory and membrane proteins, a minor but potentially disruptive population the rapid disposal of which is critical to maintaining cellular homeostasis.

Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein.

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

Reduced translocation of nascent prion protein during ER stress contributes to neurodegeneration.

During acute stress in the endoplasmic reticulum (ER), mammalian prion protein (PrP) is temporarily prevented from translocation into the ER and instead routed directly for cytosolic degradation. This "pre-emptive" quality control (pQC) system benefits cells by minimizing PrP aggregation in the secretory pathway during ER stress. However, the potential toxicity of cytosolic PrP raised the possibility that persistent pQC of PrP contributes to neurodegeneration in prion diseases. Here, we find evidence of ER stress and decreased translocation of nascent PrP during prion infection. Transgenic mice expressing a PrP variant with reduced translocation at levels expected during ER stress was sufficient to cause several mild age-dependent clinical and histological manifestations of PrP-mediated neurodegeneration. Thus, an ordinarily adaptive quality-control pathway can be contextually detrimental over long time periods. We propose that one mechanism of prion-mediated neurodegeneration involves an indirect ER stress-dependent effect on nascent PrP biosynthesis and metabolism.

Substrate-specific translocational attenuation during ER stress defines a pre-emptive quality control pathway.

Eukaryotic proteins entering the secretory pathway are translocated into the ER by signal sequences that vary widely in primary structure. We now provide a functional rationale for this long-observed sequence diversity by demonstrating that differences among signals facilitate substrate-selective modulation of protein translocation. We find that during acute ER stress, translocation of secretory and membrane proteins is rapidly and transiently attenuated in a signal sequence-selective manner. Their cotranslational rerouting to the cytosol for degradation reduces the burden of misfolded substrates entering the ER and represents a pathway for pre-emptive quality control (pQC). Bypassing the pQC pathway for the prion protein increases its rate of aggregation in the ER lumen during prolonged stress and renders cells less capable of viable recovery. Conversely, pharmacologically augmenting pQC during ER stress proved protective. Thus, protein translocation is a physiologically regulated process that is utilized for pQC as part of the ER stress response.

Protection from cytosolic prion protein toxicity by modulation of protein translocation.

Failure to promptly dispose of undesirable proteins is associated with numerous diseases. In the case of cellular prion protein (PrP), inhibition of the proteasome pathway can generate a highly aggregation-prone, cytotoxic form of PrP implicated in neurodegeneration. However, the predominant mechanisms that result in delivery of PrP, ordinarily targeted to the secretory pathway, to cytosolic proteasomes have been unclear. By accurately measuring the in vivo fidelity of protein translocation into the endoplasmic reticulum (ER), we reveal a slight inefficiency in PrP signal sequence function that generates proteasomally degraded cytosolic PrP. Attenuating this source of cytosolic PrP completely eliminates the dependence on proteasomes for PrP degradation. This allows cells to tolerate both higher expression levels and decreased proteasomal capacity without succumbing to the adverse consequences of misfolded PrP. Thus, the generation of potentially toxic cytosolic PrP is controlled primarily during its initial translocation into the ER. These results suggest that a substantial proportion of the cell's constitutive proteasomal burden may consist of proteins that, like PrP, fail to cotranslationally enter the secretory pathway with high fidelity.

Prion protein trafficking and the development of neurodegeneration.

The prion protein (PrP) is involved in causing a group of diverse transmissible, heritable and sporadically occurring neurodegenerative diseases. Although the identity, nature and replication of the transmissible agent have been intensely studied for decades, the cellular events underlying neuronal dysfunction and death have received comparatively little attention. Recent studies examining the occurrence and consequences of inappropriate cytoplasmic expression of the normally cell-surface PrP underscore an emerging role for PrP trafficking in prion disease pathogenesis.

Role for RhoB and PRK in the suppression of epithelial cell transformation by farnesyltransferase inhibitors.

Recent genetic investigations have established that RhoB gain-of-function is sufficient to mediate the antitransforming effects of farnesyltransferase inhibitors (FTIs) in H-Ras-transformed fibroblast systems. In this study, we addressed the breadth and mechanism of RhoB action in epithelial cells transformed by oncoproteins which are themselves insensitive to FTI inactivation. Rat intestinal epithelial (RIE) cells transformed by activated K-Ras or Rac1 were highly sensitive to FTI-induced actin reorganization and growth inhibition, despite the inability of FTI to block prenylation of either K-Ras or Rac1. Ectopic expression of the geranylgeranylated RhoB isoform elicited in cells by FTI treatment phenocopied these effects. Analysis of RhoB effector domain mutants pointed to a role for PRK, a Rho effector kinase implicated in the physiological function of RhoB in intracellular receptor trafficking, and these findings were supported further by experiments in a fibroblast system. We propose that FTIs recruit the antioncogenic RhoB protein in the guise of RhoB-GG to interfere with signaling by pro-oncogenic Rho proteins, possibly by sequestering common exchange factors or effectors such as PRK that are important for cell transformation.