Previous studies have shown that the oncogenic HPV E6 proteins form a complex with the human homologue of the Drosophila tumour suppressor protein, discs large (Dlg). This is mediated by the carboxy terminus of the E6 proteins and involves recognition of at least one PDZ domain of Dlg. This region of E6 is not conserved amongst E6 proteins from the low risk papillomavirus types and, hence, binding of HPV E6 proteins to Dlg correlates with the oncogenic potential of these viruses. We have performed studies to investigate the consequences of the interaction between E6 and Dlg. Mutational analysis of both the HPV18 E6 and Dlg proteins has further de®ned the regions of E6 and Dlg necessary for complex formation. Strikingly, co-expression of wild type HPV18 E6 with Dlg in vitro or in vivo results in a dramatic decrease in the amount of Dlg protein, whereas mutants of E6 which fail to complex with Dlg have minimal e ect on Dlg protein levels. The oncogenic HPV16 E6 also decreased the Dlg levels, but this was not observed with the low risk HPV11 E6 protein. Moreover, a region within the ®rst 544 amino acids of Dlg containing the three PDZ domains confers susceptibility to E6 mediated degradation. Finally, treatment of cells with a proteasome inhibitor overrides the capacity of E6 to degrade Dlg. These results demonstrate that Dlg is targeted by high risk HPV E6 proteins for proteasome mediated degradation.

Osteoclasts as effector cells in skeletal malignanciesSkeletal complications represent frequent and significant events in patients with multiple myeloma, and include osteolytic lesions, pathologic fractures, neurologic symptoms (pain, paralysis), and profound hypercalcemia. 1,2 At the cellular level, these complications are due to an excessive growth of malignant myeloma cells within the bone marrow microenvironment and their interactions with osteoblastic and osteoclastic lineage cells. 1,3,4 A consistent histologic finding in myeloma bone disease is enhanced and uncontrolled osteoclastic bone resorption adjacent to areas of plasma cell infiltrates. 2 Moreover, antiresorptive drugs that inhibit osteoclastic functions such as bisphosphonates are successfully used in patients with myeloma bone disease, indicating that osteoclasts are essential mediators in the pathogenesis of myeloma bone disease. 5 In the past 5 years, an essential cytokine system for osteoclast biology has been characterized. 6,7 This system consists of a ligand, receptor activator of NF-B ligand (RANKL), 8,9 a cellular receptor, RANK, 8,10 and a soluble decoy receptor, osteoprotegerin (OPG). 11 While RANKL stimulates several aspects of osteoclast function, thus enhancing bone resorption, OPG blocks RANKL, and prevents bone resorption. 9,12 Abnormalities of this system have been implicated in the pathogenesis of various skeletal diseases characterized by enhanced osteoclastic activity and increased bone resorption, including osteolytic metastasis and tumor-associated hypercalcemia. 13 RANKL and OPG in bone cell biologyOsteoclasts are derived from macrophagic/monocytic lineage cells and represent differentiated, multinucleated cells specialized in resorbing bone. 6,7 Recently, the essential cytokines of osteoclast biology have been identified and extensively characterized. Osteoclastic lineage cells express RANK, a member of the tumor necrosis factor receptor superfamily. 8,10 Following activation of RANK by its ligand, RANKL, differentiation, proliferation, and survival of preosteoclast is enhanced, osteoclastic fusion and activation is promoted, and osteoclastic apoptosis is suppressed, resulting in a marked increase of the number and activity of osteoclasts. 9,12 RANKL is mainly produced by osteoblastic lineage cells, 14 immune cells, 8,15 and some cancer cells. 16,17 This provides the cellular and molecular basis for osteoblast-osteoclast cross-talks, which are crucial for an orderly sequence of bone resorption and formation during bone remodeling. 14 However, RANKL production by immune and cancer cells also forms the basis of skeletal complications of inflammatory and malignant diseases, because activated T cells and cancer cells are able to directly activate RANK on osteoclasts by virtue of expressing RANKL. 4,7 The potent stimulatory effects of RANKL on RANK are counteracted by a safeguard mechanism. Many cell types-in the bone marrow microenvironment, mainly osteoblastic lineage cells-secrete OPG, which acts as a decoy receptor and bloc...

Ubiquitin ligases are generally assumed to play a major role in substrate recognition and thus provide specificity to a particular ubiquitin modification system. The multicopy maintenance protein (Mcm) 7 subunit of the replication licensing factor-M was identified as a substrate of the E3-ubiquitin ligase/E6-AP by its interaction with human papillomavirus-18E6. Mcm7 is ubiquitinated in vivo in both an E6-AP-dependent and -independent manner. E6-AP functions in these reactions independently of the viral oncogene E6. We show that recognition of Mcm7 by E6-AP is mediated by a homotypic interaction motif present in both proteins, called the L2G box. These findings served as the basis for the definition of substrate specificity for E6-AP. A small cluster of proteins whose function is intimately associated with the control of cell growth and/or proliferation contains the L2G box and is thereby implicated in an E6-AP and, by default, HPV-E6-dependent ubiquitination pathway.Selective proteolysis represents a fast and irreversible way for the control of the regulation of transition states in biology and is commonly employed from bacteriophages to human cells (1). Post-translational modification of a lysine residue from an acceptor protein by ⑀-amidation with the C-terminal glycine residue of a poly-ubiquitin chain serves as a signal for selective proteolysis by the 26 S proteasome (2). This modification is a particularly effective form of regulation for many cellular control processes where a certain unidirectional and irreversible sequential order of events is crucial for the fidelity of a system, such as the regulation of S-phase entry or the anaphase cell cycle transition (3). Ubiquitin is transferred onto substrate proteins by an enzymatic cascade (4). The ubiquitin is first activated as a thiol ester on a ubiquitin-activating enzyme in an ATP-dependent reaction, and the ubiquitin thiols are then transferred to the ubiquitin-conjugating E2 1 enzyme and finally ligated to the target protein in concert with the E3 specificity factors. These E3 components provide substrate recognition and are thus generally considered to give specificity to the ubiquitin-mediated proteolysis system. E6-AP in association with the oncogenic E6 proteins from the human papillomavirus (HPV) resembles an E3 enzyme in that it targets the cellular tumor suppressor p53 for ubiquitin-mediated degradation (5-7). In contrast with other E3 proteins, such as the anaphasepromoting complex (3), or the S-phase entry specific SKP1, Cdc53, F box protein complex (8, 9), E6-AP functions not only as an adapter between a ubiquitin acceptor substrate protein and the E2 enzyme but has intrinsic ubiquitin ligase activity. Thus E6-AP is a thiol ubiquitin acceptor from its E2 enzyme, selects substrate, and serves as the ultimate ubiquitin donor that directly couples activated ubiquitin to target proteins (10). Besides its E6-associated function, E6-AP mutations have been linked with Angelman syndrome, and this serves as the first example of a genetic disorder asso...

We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.

Age is a well-known risk factor in trauma patients. The aim of the present study was to define the age-dependent cut-off for increasing mortality in multiple injured patients. Pre-existing medical conditions in older age and impaired age-dependent physiologic reserve contributing to a worse outcome in multiple injured elderly patients are discussed as reasons for increased mortality. A retrospective clinical study of a statewide trauma data set from 1993 through 2000 included 5375 patients with an Injury Severity Score (ISS) > or = 16 who were stratified by age. The ISS and Abbreviated Injury Score (AIS) quantified the injury severity. Outcome measures were mortality, shock, multiple organ failure, and severe head injury. Mortality in this series increased beginning at age 56 years, and that increase was independent of the ISS. The mortality rate increased from 7.3% (patients 46-55 years of age) to 13.0% (patients ages 56-65 years) in patients with ISS 16-24; from 23.8% to 32.1% in those with ISS 25-50; and from 62.2% to 82.1% in those with ISS 51-75 (P < or = 0.05). Severe traumatic brain injury (sTBI) was the most frequent cause of death, with a significant peak in patients older than 75 years. The incidence of lethal multiple organ failure increased significantly beginning at age 56 years (P < or = 0.05), but it showed no further increase in patients aged 76 years or older. In contrast, the incidence of lethal shock showed a significant increase from age 76 years (P < or = 0.05), but not at age 56 years. However, from age 56 years, mortality increased significantly in patients who sustained multiple trauma-an increase that was independent of trauma severity.