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Carcinogenic progression of high-risk human papillomavirus (HPV)-infected lesions is a rare consequence of long-term persistent infections. HPV-associated cancers generally represent nonproductive infections, that is, viral proteins are expressed but no infectious virus is produced. Hence, there is no evolutionary advantage for a given HPV to cause cancer. The carcinogenic activities of high-risk HPV E6 and E7 proteins represent activities that are necessary for the viral life cycle. As a consequence of the molecular architecture of squamous epithelia, high-risk HPVs evolved to establish and maintain persistent infections of mitotically active, basal epithelial cells, while completing their replicative life cycle in terminally differentiated, growth-arrested epithelial cells. The strategies that these viruses had to develop in order to reprogram the cells and to subvert cellular surveillance mechanisms that would eliminate HPV-infected cells fundamentally alter the biological identity of the infected host cell. HPVs subvert transcriptional programs, which affect turnover of cellular proteins, induce significant changes to the epigenome and cause genomic instability. As a consequence, HPV-infected cells acquire unique vulnerabilities that may be harnessed therapeutically.

Flavonoids are a family of polyphenolic photochemical that are naturally found in plants. Flavonoids have been widely studied due to the curiosity of scientists about the implementation of nature to human health. These experimental and epidemiological studies showed that flavonoids have beneficial effects such as antioxidation, antiinflammation, antiplatelet, antiallergic, antiaging, antidiabetic, and anticarcinogenic activity. They can also interact with and regulate cellular proteins, transcription factors, and signaling enzymes at the molecular level. Cancer is one of the most common problems in human health and millions of people die due to cancer every year. Due to the lack of completely efficient therapies for most cancer patients, new approaches should be discovered. Flavonoids have been investigated and many studies have confirmed their importance as a therapy option in many diseases. In this chapter we review the use of flavonoids as therapeutical agents and prevention for cancer, including the recent studies as well as future perspectives in the field.

Protein homeostasis is key for life; cells must have a complete set of proteins within a defined range of concentrations and with a functional native conformation. Molecular chaperones play a central role in maintaining cellular proteins in their correct conformation by assisting in their folding or in their elimination by proteases if they cannot be folded. This article presents the principal chaperones that occur in the three domains of life.

In order to measure protein half-life by pulse-chase analysis, cells are incubated with labeled precursors, [(35)S]-methionine and [(35)S]-cysteine, for a short time (pulse), after which they are washed from the labeled compound and incubated with excess cold precursor (chase). The decay of the amount of protein labeled during the pulse period is measured during the chase with cold precursor.

Glucocorticoids (GCs) affect virtually every cell type and tissue in the human body, as can be deduced from the broad range of side effects that can occur upon GC treatment. The prime target for GCs is the glucocorticoid receptor (GR), and while many more cellular proteins are involved in the response to GCs, the receptor itself remains today the best-investigated source of variation in the response to GCs.

The experimental problems associated with in vivo studies of essential proteins or integral membrane proteins have triggered geneticists to generate novel approaches that have often led to insights of general relevance (Shuman and Silhavy, 2003). In order to extend the experimental portfolio, we developed target-directed proteolysis (TDP), an in vivo method allowing structural and functional characterization of target proteins in living cells. TDP is based on the activity of the highly sequence-specific NIa protease from tobacco etch virus. When its recognition site of seven residues is engineered into target proteins and NIa protease is expressed under tight promoter control, substrates can be conditionally processed while other cellular proteins remain unaffected. Applications include conditional inactivation as well as functional characterization of target proteins.

Publisher Summary This chapter examines the epidemiological data suggesting a causal relationship between human papillomavirus (HPV) and head and neck squamous cell carcinoma (HNSCC), along with a review of the transforming interaction between host cellular proteins and HPV viral proteins. Although much circumstantial evidence currently exists, the literature still lacks unequivocal evidence linking HPV with HNSCC. When all anatomic sites are considered, the prevalence of HPV in HNSCC does not appear to be higher than the prevalence of HPV in clinically normal mucosa. Although HPVs have demonstrated the ability to induce oncogenic transformation of cells in vitro, the causal relationship between HPV and HNSCC remains controversial. However, it is difficult to ignore the highly preferential association of HPV with tonsillar carcinoma, as well as the finding of high-risk HPV type 16 in these tumors. Epidemiological findings also lend further support to a causal relationship between HPV and HNSCC, particularly for oropharyngeal tumors. An etiological association between HPV and oral cancer implies that preventive interventions can be designed.

Publisher Summary This chapter discusses the mechanism of catalysis with particular focus on the enzymology of addition of the farnesyl isoprenoid to cellular proteins. Efforts to design selective cell-active inhibitors of the enzyme for evaluation as anticancer agents have been successful; the acquisition of mechanistic and structural information on protein farnesyltransferase (FTase) has greatly aided these drug discovery programs. Remaining questions on this enzyme are primarily centered on the definition of the precise chemical mechanism of the reaction and the structural basis for the ability of the enzyme to discriminate among similar protein substrates. While the close similarities in both structure and function of FTase and geranylgeranyltransferase type I (GGTase-I) make it likely that both enzymes use a similar catalytic mechanism, it will still be important to conduct detailed studies with GGTase-I to determine whether there are important differences among these enzymes.

Publisher Summary This chapter discusses chromophore-assisted laser inactivation (CALI) as a means of addressing functional interactions in biology with a high degree of spatial and temporal resolution. CALI is analogous to cellular laser ablation but the damage is localized to a protein of interest by covalently attaching the chromophore, malachite green, to an antibody that specifically binds to that protein. The laser energy is targeted to effectively inactivate the bound protein while unbound proteins are unaffected. This perturbation of function occurs at the time of laser irradiation and only within the diameter of the laser spot. This opens up the possibility of perturbing the function of a given protein in a spatially and temporally controlled manner. The chapter describes the mechanism of CALI and shows that the CALI is spatially restricted to within 34Ǻ, of the dye molecules and that the mechanism of CALI is mediated by photogenerated hydroxyl radicals. The high reactivity and very short lifetime of this radical species is responsible for the spatial specificity of CALI.

Publisher Summary S-Thiolated proteins—mixed disulfides of proteins and low molecular weight thiols—are very early products of protein oxidation during oxidative stress, occurring within seconds after the generation of oxygen radicals. As a result, assessment of the extent and specificity of this process during oxidative stress is one of the best measures of the primary effects of oxygen radical generation on intact cells. The development of methods for measuring the S-thiolation status of individual proteins, eventually studying organs of intact animals and even humans, is essential for a more complete understanding of the role of oxidative stress in human disease. This chapter introduces a method to quantify the S-thiolation status of mixed cellular proteins in intact cells and second method to determine the activity of the cellular reductive processes—namely, dethiolases. This method tests the effects of toxic materials on protein S-thiolation. Protein dethiolation can be measured with S-thiolated substrate proteins containing a radioactive low molecular weight thiol.

Publisher Summary This chapter discusses analysis of G-protein α and βγ subunits by in vitro translation. The analysis of G-protein subunits that have been modified by point mutation, deletion, or formation of chimeras requires that the altered protein be expressed. Cell-free transcription and translation of modified cDNAs offer certain advantages and disadvantages compared to various cellular systems. An important disadvantage is that cell-free translation yields only a small amount of synthesized protein that is generated in a concentrated mixture of many cellular proteins. It would, therefore, be very difficult to purify the small amount of specific protein from the very large amount of cellular components. This disadvantage is counterbalanced by the ability to radiolabel the specific protein selectively. Because only specific mRNA is added to the in vitro translation mixture, the desired protein usually represents the only radioactive band produced. Thus, if methods exist to evaluate the structure and function of the radioactive protein, without purifying it from the nonradioactive proteins present, then in vitro translation offers a rapid and attractive way of screening the consequences of modifications introduced into proteins. The chapter discusses techniques that can be used to assay the function of G-protein α, β, and γ subunits that have been synthesized in vitro. The modifications that have been made to standard in vitro transcription-translation systems are reviewed in order to enhance the yield of G-protein subunits.

The cellular proteins of Saccharomyces cerevisiae have been labelled continuously during vegetative growth using 35SO42-. During subsequent sporulation in sulphur-free sporulation medium the proteins were analysed using two-dimensional polyacrylamide gel electrophoresis. Previously it has been shown that about half of the changes found in the detectable polypeptides were specific to the sporulation process. The changes may be due to either de novo synthesis or modification of existing proteins. The present work reports that these changes occur at characteristic times throughout sporulation with some detectable very soon after transfer to sporulation medium. The bulk of the changes seem to occur within the first eight hours of sporulation. KEYWORDS Sporulation specific proteins;, temporal sequence;, gene expression.

The induction by Interferons of the synthesis of specific cellular proteins can be applied to investigate whether cells have been exposed to IFNs. For both the (2′-5′) oligo A synthetase and the HLA cell surface antigens, induction by IFNs was demonstrated at the level of mRNA, and the specificity for various IFNs was studied. Clinical applications of the synthetase assay in peripheral white blood cells are described.

A study of the kinetics of sulfur incorporation into the molecule of β-galactosidase during the induced synthesis of this enzyme in E. coli brings proof that the enzyme-protein is synthesized entirely de novo without any appreciable participation of materials coming from other cellular proteins. Furthermore, there is no measurable renewal of β-galactosidase sulfur in growing cells whether or not the enzyme is being synthesized. The induced synthesis of β-galactosidase appears as a virtually irreversible process. The bulk of the other cellular proteins in E. coli are equally stable and do not undergo any appreciable degradation and resynthesis during growth. The apparent contradiction between these results and the generally accepted concepts regarding the dynamic state of intracellular proteins is discussed.

Publisher Summary This chapter discusses the cellular protein - mixed disulfides. Potential regulation of metabolic reactions by reversible oxidation- reduction of accessible enzyme thiols has renewed interest in the redox state of cellular proteins as a function of different metabolic states. While the oxidation of thiols can lead to the formation of protein-mixed disulfides as well as intramolecular disulfides, the latter are difficult to detect in crude tissue extracts. Changes in the concentration of protein-mixed di-sulfides are, at least in principle, more readily accessible to analysis, and a number of methods for their estimation in animal tissues have been described. The identification and analysis of tissue components bound to proteins by disulfide bonds is also critical. The current methods for the determination of cellular protein - mixed disulfides are described. Low molecular weight thiols bound to proteins by disulfide bonds can be liberated either by oxidation or reduction, and the procedures for both methods are described.

Publisher Summary This chapter describes methodology for detection and quantitation of these growth factor-modulated proteins. The system that has been extensively studied is platelet-derived growth factor (PDGF)-modulated protein synthesis by quiescent BALB/c 3T3 cells. The principles utilized should be applicable to a variety of growth factor-modulated proteins, whether synthesized during growth and/or differentiation. Perhaps the most generally applicable method is the use of density-arrested monolayers. Quiescent monolayers of BALB/c 3T3 cells are arrested in G 0 /G 1 phase. They display a replicative response to a variety of growth factors and selectively synthesize certain mRNAs and proteins. In addition, certain proteins are selectively synthesized—that is, synthesized at a greater rate than the bulk of cellular proteins. The preferential synthesis of some of these proteins occurs long before resting cells become committed to synthesize DNA and is growth factor specific.