The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) The degradation of short-lived proteins is a complex and highly regulated process (21,29). In eukaryotes, the primary mechanism of selective degradation involves ligation of the C terminus of ubiquitin to E-amino groups of lysine residues within the proteolytic substrate. Coupling of ubiquitin to proteolytic substrates is mediated by a family of ubiquitinconjugating enzymes, which has at least 12 distinct members in Saccharomyces cerevisiae (35,49). These enzymes are required for a wide range of cellular functions, including cell cycle control (35), DNA repair (35), peroxisome biogenesis (57), and heavy metal resistance (37). In the case of higher eukaryotes, ubiquitination has been implicated in the degradation of mitotic cyclins (25), p53 (50), c-mos, c-fos, and other regulators of growth and the cell cycle (9, 41).Ubiquitination targets proteins for degradation by the 26S protease. This particle contains over 20 distinct subunits, including multiple peptidase and ATPase activities and a deubiquitinating activity (19,24,45). The mechanisms by which these activities are coordinated and controlled by substrate ubiquitination are unknown. However, in vitro experiments have indicated that the efficacy of ubiquitination in signaling degradation is a function of the multiplicity and arrangement of ubiquitin groups bound to the substrate (8,21,26). Substrates of the N-end rule pathway, such as Arg-3-galactosidase (P-Gal), are modified in rabbit reticulocyte extracts by a multiubiquitin chain with ubiquitin-ubiquitin linkages at 54). Both multiubiquitination and degradation of Arg-3-Gal were blocked when UbK48R was substituted for wild-type ubiquitin, suggesting that degradation requires multiubiquitin chain formation. However, there may be substrate-to-substrate differences in the degree to which * Corresponding author. Phone: (617) 432-1144. degradation is inhibited by preventing Lys-48 chain synthesis (19,28,31).One possible basis for this variation is the existence of alternative forms of multiubiquitin chains. Lys-63 of ubiquitin can apparently be used as a multiubiquitination site on endogenous substrates in vivo (52). In the present work, a failure to form Lys-48 chains was seen to inhibit various processes despite the continued presence of Lys-63 as a potential compensatory attachment site. The phenotypic effects of preventing Lys-48 chain synthesis are more dramatic than those seen with Lys-63, consistent with the view that obligatory Lys-48 chain formation is a dominant mode of degradative signaling in the ubiquitin pathway. MATERUILS AND METHODS...

Adenoviral vectors have been explored as vaccine agents for a range of infectious diseases, and their ability to induce a potent and balanced immune response made them logical candidates to apply to the COVID-19 pandemic. The unique molecular characteristics of these vectors enabled the rapid development of vaccines with advanced designs capable of overcoming the biological challenges faced by early adenoviral vector systems. These successes and the urgency of the COVID-19 situation have resulted in a flurry of candidate adenoviral vector vaccines for COVID-19 from both academia and industry. These vaccines represent some of the lead candidates currently supported by Operation Warp Speed and other government agencies for rapid translational development. This review details adenoviral vector COVID-19 vaccines currently in human clinical trials and provides an overview of the new technologies employed in their design. As these vaccines have formed a cornerstone of the COVID-19 global vaccination campaign, this review provides a full consideration of the impact and development of this emerging platform.

The combination of cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cells and animal models. In this study, we show that concurrent addition of prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV) was less efficacious than sequential treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. If cells were incubated for 24 hours with 5-FC followed by a 24-hour GCV treatment, GCV triphosphate levels were 2-fold higher, incorporation of GCV monophosphate into DNA was 2.5-fold higher, and growth inhibition was increased 4-fold compared with simultaneous treatment. As expected, cellular dTTP levels were reduced during the 5-FC preincubation. However, dGTP pools also declined parallel to the dTTP decrease. Similar results were obtained when 5-fluorouracil or 5-fluoro-2V-deoxyuridine was used instead of CD/5-FC. These data allowed us to propose a novel hypothesis for the synergistic interaction between CD/ 5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC-mediated reduction of dTTP results in a concurrent decrease of dGTP due to allosteric regulation of ribonucleotide reductase. Because dGTP is the endogenous competitor of GCV triphosphate, depleted dGTP at the time of GCV addition results in increased GCV in DNA and cell kill. In fact, addition of deoxyguanosine during the 5-FC incubation reverses the dGTP depletion, reduces the amount of GCV monophosphate incorporated into DNA, and prevents the CD/5-FC-mediated enhancement of HSV-TK/GCV cytotoxicity. Understanding this mechanistic interaction may help recognize better strategies for creating more efficacious clinical protocols. (Cancer Res 2006; 66(6): 3230-7)

The two human colon carcinoma cell lines HT-29 and SW620, which stably express herpes simplex virus thymidine kinase (HSV-TK), are sensitized to the cytotoxic effects of the antiviral drug ganciclovir (GCV). Compared with HT-29 cells, SW620 cells were more sensitive to lower GCV concentrations (<1 microM), accumulated GCV triphosphate more rapidly, and incorporated higher levels of GCV into DNA. Following a 24-hr exposure to 10 microM GCV, bystander killing was as much as sixfold greater in SW620 cells than HT-29 cells. This bystander effect was dependent on the level of HSV-TK expression, the number of cells expressing HSV-TK, and the overall confluency of the cells. However, bystander killing did not correlate with gap junctional intercellular communication as determined by microinjection of Lucifer Yellow fluorescent dye. SW620 cells were coupled to <3% adjacent cells (compared with >50% for HT-29 cells), but were still able to transfer phosphorylated GCV to bystander cells as soon as 4 hr after drug was added. These results emphasize the importance of cell-specific metabolism in HSV-TK/GCV-mediated cytotoxicity and may suggest a novel mechanism for bystander killing.

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.