Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest.

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.

Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes.

In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE-1 proton regulatory site. Annulment of the E7-induced cytoplasmic alkalinization by specific inhibition of the NHE-1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum-independent growth, anchorage-independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F-mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformation-dependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE-1, DMA, delayed the development of HPV16-keratinocyte tumors. Our data confirm that activation of the NHE-1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.

Mmf1p, a novel yeast mitochondrial protein conserved throughout evolution and involved in maintenance of the mitochondrial genome.

A novel protein family (p14.5, or YERO57c/YJGFc) highly conserved throughout evolution has recently been identified. The biological role of these proteins is not yet well characterized. Two members of the p14.5 family are present in the yeast Saccharomyces cerevisiae. In this study, we have characterized some of the biological functions of the two yeast proteins. Mmf1p is a mitochondrial matrix factor, and homologous Mmf1p factor (Hmf1p) copurifies with the soluble cytoplasmic fraction. Deltammf1 cells lose mitochondrial DNA (mtDNA) and have a decreased growth rate, while Deltahmf1 cells do not display any visible phenotype. Furthermore, we demonstrate by genetic analysis that Mmf1p does not play a direct role in replication and segregation of the mtDNA. rho(+) Deltammf1 haploid cells can be obtained when tetrads are directly dissected on medium containing a nonfermentable carbon source. Our data also indicate that Mmf1p and Hmf1p have similar biological functions in different subcellular compartments. Hmf1p, when fused with the Mmf1p leader peptide, is transported into mitochondria and is able to functionally replace Mmf1p. Moreover, we show that homologous mammalian proteins are functionally related to Mmf1p. Human p14.5 localizes in yeast mitochondria and rescues the Deltammf1-associated phenotypes. In addition, fractionation of rat liver mitochondria showed that rat p14.5, like Mmf1p, is a soluble protein of the matrix. Our study identifies a biological function for Mmf1p and furthermore indicates that this function is conserved between members of the p14.5 family.