isolation-human-hepatocytes-two-step-collagenase-perfusion

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Grosshadern Hospital, Munich

Zta, the product of the BZLF1 gene carried by Epstein-Barr virus (EBV), is crucial for reactivation of EBV from latency. Zta is a member of the bZIP family of transcription factors, and in common with many of these, Zta possesses a conserved cysteine residue in its basic region (C189) and a further cysteine residue in its ZIP region (C222). We demonstrate that C189 is required to reactivate EBV from latency but C222 is not and that this single amino acid affects two independent functions of Zta, (i) binding to a Zta-responsive site and (ii) manipulating the cell cycle.

Mutation of a single amino acid residue in the basic region of the Epstein-Barr virus (EBV) lytic cycle switch protein Zta (BZLF1) prevents reactivation of EBV from latency.

Many viruses, with distinct replication strategies, activate DNA-damage response pathways, including the lentivirus human immunodeficiency virus (HIV) and the DNA viruses Epstein-Barr virus (EBV), herpes simplex virus 1, adenovirus and SV40. DNA-damage response pathways involving DNA-dependent protein kinase, ataxia-telengiectasia mutated (ATM) and 'ataxia-telengiectasia and Rad3-related' (ATR) have all been implicated. This review focuses on the effects of HIV and EBV replication on DNA repair pathways. It has been suggested that activation of cellular DNA repair and recombination enzymes is beneficial for viral replication, as illustrated by the ability of suppressors of the ATM and ATR family to inhibit HIV replication. However, activation of DNA-damage response pathways can also promote apoptosis. Viruses can tailor the cellular response by suppressing downstream signalling from DNA-damage sensors, as exemplified by EBV. New small-molecule inhibitors of the DNA-damage response pathways could therefore be of value to treat viral infections.

DNA-damage response pathways triggered by viral replication.

The Epstein-Barr virus transcription factor Zta (encoded by BZLF1) is a bZIP protein containing an alpha-helical coiled-coil homodimerization motif (zipper). The Zta zipper forms less-stable dimers than other bZIP proteins, and an adjacent region (CT) interacts with the zipper to form a novel structure that is proposed to strengthen the dimer. Here we question the role of the CT region for Zta function. Cross-linking experiments demonstrate that the entire CT region lies adjacent to the zipper. Detailed analyses of Zta truncation mutations identify an involvement of the proximal CT region (221 to 230) in dimer formation with a further contribution from the distal region (236 to 243). Biophysical analyses reveal that residues 221 to 230 enhance the stability of the coiled coil. The ability of the Zta truncation mutants to interact with three Zta-binding sites also requires the proximal CT region. Fine mapping of DNA-binding requirements highlighted the contribution of these amino acids for Zta function. Thus, the proximal part of the CT region is required to aid the dimerization of Zta and thereby its DNA-binding ability. In contrast, although the distal part of the CT region aids dimerization, it promotes only a modest increase in DNA binding. To probe this further, we defined the contribution from the CT region for Zta to transactivate a promoter embedded within the viral genome. From this we conclude that the proximal part of the CT region is absolutely required, whereas the distal part is dispensable.

Atypical bZIP domain of viral transcription factor contributes to stability of dimer formation and transcriptional function.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

EBV (Epstein-Barr virus) alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and in the switch to the lytic cycle this epigenetic silencing is overturned. A key event is the activation of the viral protein Zta with three ZREs (Zta-response elements) from the BRLF1 promoter (referred to as Rp). Two of these ZREs contain CpG motifs and are methylated in the latent genome. Biochemical analyses and molecular modelling of Zta bound to methylated RpZRE3 indicate the precise contacts made between a serine and a cysteine residue of Zta with methyl cytosines. A single point mutant of Zta, C189S, is defective in binding to the methylated ZREs both in vitro and in vivo. This was used to probe the functional relevance of the interaction. ZtaC189S was not able to activate Rp in a B-cell line, demonstrating the relevance of the interaction with methylated ZREs. This demonstrates that Zta plays a role in overturning the epigenetic control of viral latency.

The reversal of epigenetic silencing of the EBV genome is regulated by viral bZIP protein.

Epstein-Barr virus (EBV; human herpesvirus 4) poses major clinical problems worldwide. Following primary infection, EBV enters a form of long-lived latency in B lymphocytes, expressing few viral genes, and it persists for the lifetime of the host with sporadic bursts of viral replication. The switch between latency and replication is governed by the action of a multifunctional viral protein Zta (also called BZLF1, ZEBRA, and Z). Using a global proteomic approach, we identified a host DNA damage repair protein that specifically interacts with Zta: 53BP1. 53BP1 is intimately connected with the ATM signal transduction pathway, which is activated during EBV replication. The interaction of 53BP1 with Zta requires the C-terminal ends of both proteins. A series of Zta mutants that show a wild-type ability to perform basic functions of Zta, such as dimer formation, interaction with DNA, and the transactivation of viral genes, were shown to have lost the ability to induce the viral lytic cycle. Each of these mutants also is compromised in the C-terminal region for interaction with 53BP1. In addition, the knockdown of 53BP1 expression reduced viral replication, suggesting that the association between Zta and 53BP1 is involved in the viral replication cycle.

Functional interaction between Epstein-Barr virus replication protein Zta and host DNA damage response protein 53BP1.

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac Î²-adrenergic signaling.

Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain.

The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1Î± and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank.

RNA stability in human liver: comparison of different processing times, temperatures and methods.

High-quality biospecimens of human origin with annotated clinical and procedural data are an important tool for biomedical research, not only to map physiology, pathophysiology and aetiology but also to go beyond in translational research. This has opened a new special field of research known as 'biobanking', which focuses on how to collect, store and provide these specimens and data, and which is substantially supported by national and European funding.

Biobanking for research in surgery: are surgeons in charge for advancing translational research or mere assistants in biomaterial and data preservation?

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80Â % hepatocytes and 20Â % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4Î±, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4Î±), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Article	Year
Mutation of a single amino acid residue in the basic region of the Epstein-Barr virus (EBV) lytic cycle switch protein Zta (BZLF1) prevents reactivation of EBV from latency. Journal of virology\| PubMed ID: 16227304	2005
DNA-damage response pathways triggered by viral replication. Expert reviews in molecular medicine\| PubMed ID: 16515730	2006
Atypical bZIP domain of viral transcription factor contributes to stability of dimer formation and transcriptional function. Journal of virology\| PubMed ID: 17459922	2007
Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein. PLoS pathogens\| PubMed ID: 18369464	2008
The reversal of epigenetic silencing of the EBV genome is regulated by viral bZIP protein. Biochemical Society transactions\| PubMed ID: 18631132	2008
Functional interaction between Epstein-Barr virus replication protein Zta and host DNA damage response protein 53BP1. Journal of virology\| PubMed ID: 19656881	2009
Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain. The Journal of biological chemistry\| PubMed ID: 21965681	2011
RNA stability in human liver: comparison of different processing times, temperatures and methods. Molecular biotechnology\| PubMed ID: 22271457	2013
Biobanking for research in surgery: are surgeons in charge for advancing translational research or mere assistants in biomaterial and data preservation? Langenbeck's archives of surgery / Deutsche Gesellschaft fÃ¼r Chirurgie\| PubMed ID: 23430290	2013
Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotox... Archives of toxicology\| PubMed ID: 23974980	2013

Celine Schelcher

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