Marcel Doerflinger

BH3‐only proteins: a 20‐year stock‐take

BH3‐only proteins are the sentinels of cellular stress, and their activation commits cells to apoptosis. Since the discovery of the first BH3‐only protein BAD almost 20 years ago, at least seven more BH3‐only proteins have been identified in mammals. They are regulated by a variety of environmental stimuli or by developmental cues, and play a crucial role in cellular homeostasis. Some are considered to be tumor suppressors, and also play a significant role in other pathologies. Their non‐apoptotic functions are controversial, but there is broad consensus emerging regarding their role in apoptosis, which may help in designing better therapeutic agents for treating a variety of human diseases.

DR5 and caspase-8 are dispensable in ER stress-induced apoptosis

The endoplasmic reticulum (ER) stress response constitutes cellular reactions triggered by a wide variety of stimuli that disturb folding of proteins, often leading to apoptosis. ER stress-induced apoptotic cell death is thought to be an important contributor to many human pathological conditions. The molecular mechanism of this apoptosis process has been highly controversial with both the receptor and the mitochondrial pathways being implicated. Using knockout mouse models and RNAi-mediated gene silencing in cell lines, our group and others had demonstrated the importance of the mitochondrial apoptotic pathway in ER stress-induced cell death, particularly the role of the pro-apoptotic BH3-only BCL-2 family members, BIM and PUMA. However, a recent report suggested a central role for the death receptor, DR5, activated in a ligand-independent manner, and the initiator caspase, caspase-8, in ER stress-induced cell death. This prompted us to re-visit our previous observations and attempt to reproduce the newly published findings. Here we report that the mitochondrial apoptotic pathway, activated by BH3-only proteins, is essential for ER stress-induced cell death and that, in contrast to the previous report, DR5 as well as caspase-8 are not required for this process.

CRISPR/Cas9—The ultimate weapon to battle infectious diseases?

Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug‐resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus‐1 and hepatitis B virus.

Variola Virus F1L is a Bcl-2-Like Protein that Unlike its Vaccinia Virus Counterpart Inhibits Apoptosis Independent of Bim.

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

Role of p53 in cAMP/PKA pathway mediated apoptosis

Deregulated β-adrenoceptor/cAMP-PKA pathway is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. We recently had demonstrated that the β-adrenoceptor/cAMP-PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member BIM in tissues, such as the thymus and the heart. Induction of BIM is driven by the transcriptional co-activator CBP (CREB Binding Protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the BIM promoter site [Lee et al., Cell Death Difference 20(7):941–952 (2013)]. However since CBP is a co-factor for multiple transcription factors, BH-3 only proteins other than Bim could also contribute to this apoptosis pathway. Here we provide evidence for the involvement of p53-CBP axis in apoptosis through Puma/Noxa induction, in response to β-adrenoceptor activation. Our findings highlight the molecular complexity of pathophysiology associated with a deregulated neuro-endocrine system and for developing novel therapeutic strategies for these diseases.

Cre transgene results in global attenuation of the cAMP/PKA pathway

Use of the cre transgene in in vivo mouse models to delete a specific ‘floxed’ allele is a well-accepted method for studying the effects of spatially or temporarily regulated genes. During the course of our investigation into the effect of cyclic adenosine 3′,5′-monophosphate-dependent protein kinase A (PKA) expression on cell death, we found that cre expression either in cultured cell lines or in transgenic mice results in global changes in PKA target phosphorylation. This consequently alters gene expression profile and changes in cytokine secretion such as IL-6. These effects are dependent on its recombinase activity and can be attributed to the upregulation of specific inhibitors of PKA (PKI). These results may explain the cytotoxicity often associated with cre expression in many transgenic animals and may also explain many of the phenotypes observed in the context of Cre-mediated gene deletion. Our results may therefore influence the interpretation of data generated using the conventional cre transgenic system.

Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA

Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA

Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice

Loss of function mutations in the Prkar1a gene are the cause of most cases of Carney complex disorder. Defects in Prkar1a are thought to cause hyper-activation of PKA signalling, which drives neoplastic transformation, and Prkar1a is therefore considered to be a tumour suppressor. Here we show that loss of Prkar1a in genetically modified mice caused transcriptional activation of several proapoptotic Bcl-2 family members and thereby caused cell death. Interestingly, combined loss of Bim and Prkar1a increased colony formation of fibroblasts in culture and promoted their growth as tumours in immune-deficient mice. Apart from inducing apoptosis, systemic deletion of Prkar1a caused cachexia with muscle loss, macrophage activation and increased lipolysis as well as serum triglyceride levels. Loss of single allele of Prkar1a did not enhance tumour development in a skin cancer model, but surprisingly, when combined with the loss of Bim, caused a significant delay in tumorigenesis and this was associated with upregulation of other BH3-only proteins, PUMA and NOXA. These results show that loss of Prkar1a can only promote tumorigenesis when Prkar1a-mediated apoptosis is somehow countered.

Mycobacterium tuberculosis: Rewiring host cell signaling to promote infection

The ability of Mycobacterium tuberculosis to cause disease hinges upon successfully thwarting the innate defenses of the macrophage host cell. The pathogens trump card is its armory of virulence factors that throw normal host cell signaling into disarray. This process of subverting the macrophage begins upon entry into the cell, when M. tuberculosis actively inhibits the fusion of the bacilli‐laden phagosomes with lysosomes. The pathogen then modulates an array of host signal transduction pathways, which dampens the macrophages host‐protective cytokine response, while simultaneously adapting host cell metabolism to stimulate lipid body accumulation. Mycobacterium tuberculosis also renovates the surface of its innate host cells by altering the expression of key molecules required for full activation of the adaptive immune response. Finally, the pathogen coordinates its exit from the host cell by shifting the balance from the host‐protective apoptotic cell death program toward a lytic form of host cell death. Thus, M. tuberculosis exploits its extensive repertoire of virulence factors in order to orchestrate the infection process to facilitate its growth, dissemination, and entry into latency. This review offers critical insights into the most recent advances in our knowledge of how M. tuberculosis manipulates host cell signaling. An appreciation of such interactions between the pathogen and host is critical for guiding novel therapies and understanding the factors that lead to the development of active disease in only a subset of exposed individuals.

BH3-only proteins: the thorny end of the ER stress response

The endoplasmic reticulum (ER) is the hub of protein trafficking inside the cell. Secreted, membrane-bound and organelle-targeted proteins are all trafficked through the ER before being post-translationally modified (i.e. glycosylated and folded), making them competent for exporting. This involves coordinated functioning of chaperones, glycosyltransferases and protein disulfide isomerases. Perturbation of ER homeostasis—such as disturbance of ATP, calcium levels or change in the redox status—can affect protein folding and lead to protein aggregation and ER stress. ER stress leads to two divergent cellular responses: the unfolded protein response—where cells mitigate the stress by reducing protein load and upregulating the production of chaperones; should the cells be overwhelmed by the stress, they eventually reach a point of no return, at which point they instigate apoptosis. While the former response is necessary for developmental processes—such as plasma and dendritic cell differentiation and muscle fiber formation— apoptosis is often harmful. It is attributed to many human pathologies, such as cystic fibrosis, α1-antitrypsin deficiency, thyroglobulin deficiency and diabetes insipidus. For this reason, the apoptotic response of ER stress has been the subject of intense study for the past couple of decades. Understanding the molecular mechanism of ER stressinduced apoptosis has been a theorist’s paradise, and has been highly controversial. One of the earliest reports on the mechanism was by Nakagawa et al., who claimed that caspase-12 was the main instigator of ER stress-mediated apoptosis. Caspase-12 appeared to be localized to the ER and activated by ER stress, and genetic ablation of caspase12 in mice resulted in protection of cortical neurons from β-amyloid-induced apoptosis. However, subsequent study by Saleh et al. contradicted this on a number of levels. First, caspase-12 mouse embryonic fibroblasts (MEFs) did not offer protection against apoptosis induced by a variety of ER stress inducing agents. In addition, apart from a subgroup of the African population, most human populations do not produce functional caspase-12. The role of caspase-12 in mice and in human populations, where the functional protein is expressed, appears to be in modulating inflammatory response to infections. Lack of functional caspase-12 in humans prompted Hitomi et al. to suggest that caspase-4 was the equivalent of caspase-12 in humans and had a crucial role to play in ER stress-induced apoptosis in human cells in response to β-amyloids. However, this assertion was also discredited in subsequent studies, where both caspase-12 and caspase-4 were found not to be involved in ER stressinduced apoptosis. In cells expressing either murine caspase12 or human caspase-4, ER stress-induced apoptosis could be blocked by over expression of Bcl-XL or by a dominant negative form of caspase-9—suggesting a role for the mitochondrial pathway. There is mounting evidence for the role of BH3-only members of the Bcl-2 family in this process. While earlier studies defined a role for PUMA and NOXA in ER stressinduced apoptosis in neuronal cells and in MEFs, we had reported a role for BIM in lymphocytes, macrophages and epithelial cells. We also recently reported a role for ER stress-mediated BIM induction in lymphocytes during sepsis in mice. Thus, there appears to be a division of labor among various BH3-only proteins (i.e. PUMA largely in neuronal cells, NOXA and PUMA in fibroblasts and BIM in lymphocytes, myeloid cells as well as epithelial cells in inducing ER stress-mediated apoptosis). There was a suggestion that the BH3-only proteins BIM and PUMA were the upstream modulators of ER stress—where these two proteins could interact with IRE1α and regulate XBP-1 splicing, lymphocyte maturation and IgM secretion. However, our attempts to reproduce these results were unsuccessful. Our recent results, using a variety of gene knockout cell lines and via activated caspase-specific pull-down experiments, unequivocally show a central role for the mitochondrial pathway and BH3-only proteins in this apoptotic process. Again, the role of individual BH3-only protein appears to be cell type-specific (Figure 1). Elucidation of the precise molecular mechanism(s) of ER stress-induced apoptosis is crucial for developing therapeutics for many diseases, particularly for cancer. Hypoxic conditions in the microenvironment and increased demand for protein synthesis mean tumor cells have high levels of ER stress and evolve mechanisms to overcome the resulting apoptosis. For this reason, a great many chemotherapeutic drugs—including NSAIDs, proteasomal inhibitors and HDAC inhibitors—all work by inducing the apoptotic arm of the ER stress