Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.     

Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins.

The E6 and the E7 proteins of the oncogenic human papillomavirus types 16 and 18 can stably associate with p53 and the retinoblastoma protein, respectively. The E6-p53 interaction results in the accelerated degradation of p53 in vitro via the ubiquitin-dependent proteolysis system. In this study we demonstrate that a fusion protein consisting of the N-terminal half of the HPV-16 E7 protein and the full length HPV-16 E6 protein promotes the in vitro degradation of the retinoblastoma protein. This indicates that the property of the HPV-16 E6 protein to stimulate the degradation of p53 can be targeted to other proteins. Unlike the HPV-16 or HPV-18 E6 protein, the E6 proteins of HPV-6 and 11 do not bind to p53 and consequently do not target p53 for degradation. Analogous E7-E6 fusion proteins using the E6 proteins of HPV-6 and HPV-11, however, also have the ability to promote the degradation of the retinoblastoma protein, indicating that the property to target associated proteins for degradation is shared by the anogenital specific HPV E6 proteins.     

Transformation of dog embryo kidney cells by human herpesviruses

The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.     

Regulation of osteogenic proteins by chondrocytes

The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases.     

Understanding human T-cell-mediated immunoregulation through herpesviruses

Human herpesviruses have coevolved with humans over millions of years, and adaptation of latent infection within the cells of the immune system is a unique characteristic of many of these viruses. Following primary infection, these herpesviruses establish an asymptomatic-persistent infection in healthy individuals that is strictly controlled by virus-specific CD8(+) and CD4(+) T cells. Here, we provide a brief overview of how the human immune system interacts with these latent viruses and regulates the lifelong host-virus relationship in healthy virus carriers. Extensive studies on T-cell-mediated immune regulation over the last decade has allowed researchers to successfully translate these findings into the clinical setting to treat various herpesvirus-associated diseases in transplant patients and individuals with virus-associated malignancies. It is highly likely that these newly emerging T-cell-based therapeutic and diagnostic technologies will revolutionize the clinical management of patients with herpesvirus-associated diseases.     

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.     

Regulation of cardiac contractile proteins by phosphorylation

Several of the contractile proteins of the heart can be phosphorylated, but in studies with isolated proteins only phosphorylation of the inhibitory subunit of troponin (TnI) produces a major change in the properties of the contractile system. As TnI is phosphorylated, the concentration of calcium required for activation of contraction is increased. Phosphorylation of the tropomyosin-binding subunit of troponin (TnT) or of the light chain of myosin fails to change ATPase activity of the isolated protein system. Phosphorylation of TnI is stimulated by the beta-adrenergic system and inhibited by the cholinergic system. Maximum calcium-activated force produced by the contractile system can be increased in hyperpermeable cardiac cells by cyclic AmP (cAMP) or agents that stimulate cAMP synthesis. This change in the contractile system, which appears to be part of the physiological response to beta-adrenergic stimulation, is mediated by phosphorylation of an intermediate that then modifies the contractile system. Phosphorylation of the contractile proteins is not involved.     

Regulation of cytoskeletal dynamics by actin-monomer-binding proteins

The actin cytoskeleton is a vital component of several key cellular and developmental processes in eukaryotes. Many proteins that interact with filamentous and/or monomeric actin regulate the structure and dynamics of the actin cytoskeleton. Actin-filament-binding proteins control the nucleation, assembly, disassembly and crosslinking of actin filaments, whereas actin-monomer-binding proteins regulate the size, localization and dynamics of the large pool of unpolymerized actin in cells. In this article, we focus on recent advances in understanding how the six evolutionarily conserved actin-monomer-binding proteins - profilin, ADF/cofilin, twinfilin, Srv2/CAP, WASP/WAVE and verprolin/WIP - interact with actin monomers and regulate their incorporation into filament ends. We also present a model of how, together, these ubiquitous actin-monomer-binding proteins contribute to cytoskeletal dynamics and actin-dependent cellular processes.     

Regulation of calcium channels by RGK proteins

The RGK family of proteins, small GTPases of the Ras superfamily, are known to regulate calcium currents. It is commonly thought that this is due to an interaction with the Cavβ subunit, however, the mechanism of this inhibition is unclear. There have been conflicting reports of whether RGK proteins can affect channel trafficking or whether they reduce calcium currents by interacting with channels at the membrane. In the last year, several studies have emerged which explore the intricacies of RGK protein interaction with the channel itself and the importance of the Cavβ subunit for this interaction, in addition to providing some tantalizing suggestions for the mechanism by which RGK proteins reduce or eliminate calcium currents. In this review, we present an overview of these recent advances and suggest a model that may synthesize these latest works.     

Regulation of N-myristoyltransferase by novel inhibitor proteins

N-myristoylation ensures the proper function and intracellular trafficking of proteins. Many proteins involved in a wide variety of signaling, including cellular transformation and oncogenesis, are myristoylated. The myristoylation of proteins is catalyzed by the ubiquitously distributed eukaryotic enzyme N-myristoyltransferase (NMT). Previously, we reported that NMT activity is higher in colonic epithelial neoplasms than in normal-appearing colonic tissue and that the increase in NMT activity appears at an early stage in colonic carcinogenesis. Furthermore, we observed that NMT expression is elevated in colorectal and gallbladder carcinoma. In our laboratory, an endogenous NMT inhibitor protein (NIP71) was discovered from bovine brain that inhibited NMT activity in rat colonic tumors. Very recently we have demonstrated that the protein NIP71, which is a potential inhibitor of NMT, is homologous to heat-shock cognate protein (HSC70). In addition, we have discovered that enolase is a potent inhibitor of NMT. Further work may elucidate the role of HSC70 and/or enolase in the regulation of NMT, which may lead to the development of a gene-based therapy of colorectal cancer. The interaction of oncoproteomic and oncogenomic data sets through powerful bioinformatics will yield a comprehensive database of protein properties, which will serve as an invaluable tool for cancer researchers to understand the progress of tumorigenesis.     

Regulation of phospholipase C by G proteins

Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the G_q subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the G_q subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.     

Regulation of Sin recombinase by accessory proteins

Sin recombinase from Staphylococcus aureus acts selectively on directly repeated resH sites, assembling an intertwined synapse in which exactly three supercoils are trapped between the points of strand exchange. Resolution requires the two Sin binding sites in resH (site I, where strand exchange occurs, and site II) and a non-specific DNA-bending protein (e.g. Hbsu). We show that a single amino acid substitution in Sin (I100T) is sufficient to relax the normal requirements for site II and Hbsu. Using this hyperactive protein, and the variant recombination site resH(AT), we investigate the roles of site II and Hbsu in synapsis and strand exchange. We conclude that Sin bound at site II, and Hbsu, act together to control site I alignment and the topology of the synapse, and to stimulate strand exchange.