The microvascular endothelial cell monolayer localized at the critical interface between the blood and vessel wall has the vital functions of regulating tissue fluid balance and supplying the essential nutrients needed for the survival of the organism. The endothelial cell is an exquisite "sensor" that responds to diverse signals generated in the blood, subendothelium, and interacting cells. The endothelial cell is able to dynamically regulate its paracellular and transcellular pathways for transport of plasma proteins, solutes, and liquid. The semipermeable characteristic of the endothelium (which distinguishes it from the epithelium) is crucial for establishing the transendothelial protein gradient (the colloid osmotic gradient) required for tissue fluid homeostasis. Interendothelial junctions comprise a complex array of proteins in series with the extracellular matrix constituents and serve to limit the transport of albumin and other plasma proteins by the paracellular pathway. This pathway is highly regulated by the activation of specific extrinsic and intrinsic signaling pathways. Recent evidence has also highlighted the importance of the heretofore enigmatic transcellular pathway in mediating albumin transport via transcytosis. Caveolae, the vesicular carriers filled with receptor-bound and unbound free solutes, have been shown to shuttle between the vascular and extravascular spaces depositing their contents outside the cell. This review summarizes and analyzes the recent data from genetic, physiological, cellular, and morphological studies that have addressed the signaling mechanisms involved in the regulation of both the paracellular and transcellular transport pathways.

https://www.physiology.org/doi/pdf/10.1152/physrev.00012.2005

Signaling Mechanisms Regulating Endothelial Permeability

ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response.

Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR–PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.

Proteinase-Activated Receptors

Epigenetics is one of the most promising and expanding fields in the current biomedical research landscape. Since the inception of epigenetics in the 1940s, the discoveries regarding its implications in normal and disease biology have not stopped, compiling a vast amount of knowledge in the past decade. The field has moved from just one recognized marker, DNA methylation, to a variety of others, including a wide spectrum of histone modifications. From the methodological standpoint, the successful initial single gene candidate approaches have been complemented by the current comprehensive epigenomic approaches that allow the interrogation of genomes to search for translational applications in an unbiased manner. Most important, the discovery of mutations in the epigenetic machinery and the approval of the first epigenetic drugs for the treatment of subtypes of leukemias and lymphomas has been an eye-opener for many biomedical scientists and clinicians. Herein, we will summarize the progress in the field of cancer epigenetics research that has reached mainstream oncology in the development of new biomarkers of the disease and new pharmacological strategies.

Cancer epigenetics reaches mainstream oncology

Apoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.

/pdf/many-cuts-to-ruin-a-comprehensive-update-of-caspase-3w9xulij2x.pdf

Many cuts to ruin: a comprehensive update of caspase substrates

To say that thrombin is a multi-functional protein is rather to understate the case. It is the key enzyme involved in haemostasis, playing important roles at all levels of complexity. First, in the coagulation cascade, thrombin converts fibrinogen into fibrin, which is readily cross-linked to form a clot (reviewed in [1,2]) ; secondly, thrombin activates blood platelets, causing aggregation and secretion [3,4] ; and thirdly, thrombin can elicit mitogenic responses from vascular smooth-muscle cells [5,6]. This latter property is probably most significant in the renewal of damaged blood-vessel walls. Additionally, thrombin is able to elicit responses from cell types as diverse as macrophages [7], monocytes [8] and neutrophils [9]. Perhaps more surprisingly, it is able to regulate neurite outgrowth from cells of neuronal origin [10] and initiate resorption of bone cells [11]. All of these properties of thrombin appear to rely on its action as a serine proteinase, since modification (either chemically [12–14] or mutationally [15–17]) which destroys its proteolytic activity leads to a loss of biological activity. These crucial findings have been explained to a large extent by the recent elegant characterization of a novel, widely expressed thrombin receptor which is activated by proteolytic cleavage rather than by ligand (protein) binding [18,19]. At the same time as elucidation of the mechanism of thrombin signalling there has been a dramatic increase in our understanding of how thrombin signals are mediated within the cell. This therefore seems an appropriate time to assess our current knowledge and perhaps to try to predict areas where the greatest advances will be made in the immediate future. In this review, because of limitations in length, emphasis will be placed on recent advances, in particular on the characterization of the thrombin receptor and in the mechanisms of intracellular signalling. Whilst most studies of thrombin have concentrated on its action on cells involved in blood clotting and wound healing, it is now becoming apparent that it can modulate the growth and differentiation status of cells of neuronal origin. A consideration of recent advances in this area of study will form the third major theme of this review.

/pdf/cellular-consequences-of-thrombin-receptor-activation-48ewahfe42.pdf

Cellular consequences of thrombin-receptor activation.

It came as a considerable surprise to virologists in 1962 when John Trentin and his colleagues (Trentin et al., 1962) reported that a human adenovirus was oncogenic. At Baylor University (Houston, Texas) they had been inoculating newborn hamsters with human adenoviruses and found that adenovirus serotype 12 (Ad12) induced tumours in a high proportion of the animals injected. Other Ad serotypes (Ads) were found to be non-tumorigenic (this included Ad2, which with its closely related non-oncogenic serotype Ad5, have been the most extensively studied Ads; referred to in this review as Ad2/5). This important observation was initially greeted with some scepticism (see Gross, 1966). However, Ad12 oncogenesis was rapidly con®rmed by others (Huebner et al., 1962) and lead to more extensive in vivo and in vitro studies on the capacity of human Ads to disrupt normal cell growth control. It is not possible to review all of the literature on this subject here but there are a number of reviews that cover this subject (Gross, 1966; Gallimore et al., 1984; Williams et al., 1995). From the in vivo studies in the 1960's it soon became clear that there were indeed tumorigenic (Huebner et al., 1962; Trentin et al., 1962, Girardi et al., 1964; Pereira et al., 1965) and nontumorigenic adenoviruses (Trentin et al., 1962, 1968). As well as Ad serotype, adenovirus induced tumorigenicity was found to be dependent on virus dose (Yabe et al., 1962), host genetic constitution (Yabe et al., 1964, Yohn et al., 1965; Allison et al., 1967), age at inoculation (Yabe et al., 1962) and the host's immune status (Yohn et al., 1965; Allison et al., 1967). Around this time tissue culture studies revealed that rodent cells were susceptible to adenovirus transformation and that both tumorigenic (McBride and Wiener, 1964) and non-tumorigenic Ads (Freeman et al., 1967) induced morphological transformation from which immortal cell lines could be easily derived. Studies on immortal rodent cell lines showed that the cells transformed by non-oncogenic viruses could be tumorigenic, but only in immunocompromised hosts (Gallimore, 1972, Gallimore et al., 1977). The next logical step in adenovirus research was to identify the virus genes responsible for transformation and/or tumorigenicity. Unlike today, no restriction enzymes were readily available and there was no Southern blotting technique (Southern, 1975). Fujinaga and Green used membrane hybridization to show that adenovirus sequences were indeed retained and expressed in Ad transformed cells (Fujinaga and Green, 1970). This was followed by the ®rst attempt to utilize restriction endonucleases and Cot analysis (Pettersson and Sambrook, 1973) to map the viral sequences in an Ad2 transformed line, Ad2/8617. In the following year, a more extensive study found that a minimal region of the virus genome representing the left hand 14% was common to nine independently isolated Ad2 transformed rat embryo cell lines (Gallimore et al., 1974). This strongly suggested, but did not prove, that the Ad transforming gene resided in this region of the Ad genome. With the development of the calcium phosphate DNA transfection technique (Graham and van der Eb, 1973) and the use of de®ned Ad DNA fragments, it was convincingly shown that this region was responsible for the induction of transformation (Graham et al., 1974a,b). RNA mapping was carried out (Sharp et al., 1974; Flint et al., 1975) which de®ned the early region transcripts (reviewed by Shenk and Flint, 1991; Shenk, 1996) and later designated E1 (E1A+E1B) E2, E3 and E4 (Kitchingman et al., 1977). Viral RNA selected on speci®c Ad DNA fragments was then used in transcription/translation assays to identify the proteins expressed from these regions and their molecular weights (Lewis et al., 1976; Harter and Lewis, 1978; Halbert et al., 1979). The E1A proteins were found to have a molecular weight range from approximately 28 to 58 kDa. The next major step in our understanding of E1A was facilitated by the isolation of the now famous 293 cells, human embryo kidney cells transformed by Ad5 DNA (Graham et al., 1977). These cells expressed the Ad5 E1 transforming proteins that provided a permissive environment for replicationand transformation-defective E1 region mutants (Frost and Williams, 1978; Graham et al., 1978). Similar cell lines were developed for the isolation of Ad12 mutants (Byrd et al., 1982). The E1 regions of Ad2/5 and Ad12 Oncogene (2001) 20, 7824 ± 7835 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00

Adenovirus E1A: remodelling the host cell, a life or death experience.

Regulation of DNA-End Resection by hnRNPU-like Proteins Promotes DNA Double-Strand Break Signaling and Repair

It is clear that a number of host-cell factors facilitate virus replication and, conversely, a number of other factors possess inherent antiviral activity. Research, particularly over the last decade or so, has revealed that there is a complex inter-relationship between viral infection and the host-cell DNA-damage response and repair pathways. There is now a realization that viruses can selectively activate and/or repress specific components of these host-cell pathways in a temporally coordinated manner, in order to promote virus replication. Thus, some viruses, such as simian virus 40, require active DNA-repair pathways for optimal virus replication, whereas others, such as adenovirus, go to considerable lengths to inactivate some pathways. Although there is ever-increasing molecular insight into how viruses interact with host-cell damage pathways, the precise molecular roles of these pathways in virus life cycles is not well understood. The object of this review is to consider how DNA viruses have evolved to manage the function of three principal DNA damage-response pathways controlled by the three phosphoinositide 3-kinase (PI3K)-related protein kinases ATM, ATR and DNA-PK and to explore further how virus interactions with these pathways promote virus replication.

http://www.hixonparvo.info/DDR%26Virus%20Review%202012.pdf

DNA viruses and the cellular DNA-damage response

Recent reports suggest that an early region 1B (E1B) 55,000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (dl1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5 dl1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12 dl620 and Ad12 hr703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5 dl1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.

Andrew S. Turnell

Papers

Cellular consequences of thrombin-receptor activation.

Adenovirus E1A: remodelling the host cell, a life or death experience.

Regulation of DNA-End Resection by hnRNPU-like Proteins Promotes DNA Double-Strand Break Signaling and Repair

DNA viruses and the cellular DNA-damage response

The Replicative Capacities of Large E1B-Null Group A and Group C Adenoviruses Are Independent of Host Cell p53 Status