Comparison of the stage-dependent mitochondrial changes in response to pressure overload between the diseased right and left ventricle in the rat.

The right ventricle (RV) differs developmentally, anatomically and functionally from the left ventricle (LV). Therefore, characteristics of LV adaptation to chronic pressure overload cannot easily be extrapolated to the RV. Mitochondrial abnormalities are considered a crucial contributor in heart failure (HF), but have never been compared directly between RV and LV tissues and cardiomyocytes. To identify ventricle-specific mitochondrial molecular and functional signatures, we established rat models with two slowly developing disease stages (compensated and decompensated) in response to pulmonary artery banding (PAB) or ascending aortic banding (AOB). Genome-wide transcriptomic and proteomic analyses were used to identify differentially expressed mitochondrial genes and proteins and were accompanied by a detailed characterization of mitochondrial function and morphology. Two clearly distinguishable disease stages, which culminated in a comparable systolic impairment of the respective ventricle, were observed. Mitochondrial respiration was similarly impaired at the decompensated stage, while respiratory chain activity or mitochondrial biogenesis were more severely deteriorated in the failing LV. Bioinformatics analyses of the RNA-seq. and proteomic data sets identified specifically deregulated mitochondrial components and pathways. Although the top regulated mitochondrial genes and proteins differed between the RV and LV, the overall changes in tissue and cardiomyocyte gene expression were highly similar. In conclusion, mitochondrial dysfuntion contributes to disease progression in right and left heart failure. Ventricle-specific differences in mitochondrial gene and protein expression are mostly related to the extent of observed changes, suggesting that despite developmental, anatomical and functional differences mitochondrial adaptations to chronic pressure overload are comparable in both ventricles.

The Aurora B-controlled PP1/RepoMan complex determines the spatial and temporal distribution of mitotic H2B S6 phosphorylation.

The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.

A reductionist perspective on HIF-1α's role in cell proliferation under non-hypoxic conditions.

The role of hypoxia-inducible factor (HIF)-1α in the control of proliferation under non-hypoxic conditions has been investigated in numerous studies, but does not yield a coherent picture. Therefore, we conducted this meta-analysis of existing literature to systematically evaluate the role of HIF-1α, based on a number of inclusion and exclusion criteria. Studies analyzing non-transformed, primary cells showed a largely heterogeneous distribution of pro-proliferative, anti-proliferative or absent functions for HIF-1α, which are co-determined by several parameters, including the type and age of the cell and its localization in tissues and organs. In contrast, the analyses of tumor cells showed a predominantly pro-proliferative role of HIF-1α by cell-intrinsic and cell-extrinsic molecular mechanism not yet understood.

Open questions in the NF-κB field.

A variety of stress signals leads to activation of the inducible transcription factor NF-κB, one of the master regulators of the innate immune response. Despite a wealth of information available on the NF-κB core components and its control by different activation pathways and negative feedback loops, several levels of complexity hamper our understanding of the system. This has also contributed to the limited success of NF-κB inhibitors in the clinic and explains some of their unexpected effects. Here we consider the molecular and cellular events generating this complexity at all levels and point to a number of unresolved questions in the field. We also discuss potential future experimental and computational strategies to provide a deeper understanding of NF-κB and its coregulatory signaling networks.

Understanding and Therapeutic Targeting of the p53 Network.

Signaling networks function as highly intertwined regulatory hubs rather than linear cascades with a single endpoint [...].

A transient increase of HIF-1α during the G1 phase (G1-HIF) ensures cell survival under nutritional stress.

The family of hypoxia-inducible transcription factors (HIF) is activated to adapt cells to low oxygen conditions, but is also known to regulate some biological processes under normoxic conditions. Here we show that HIF-1α protein levels transiently increase during the G1 phase of the cell cycle (designated as G1-HIF) in an AMP-activated protein kinase (AMPK)-dependent manner. The transient elimination of G1-HIF by a degron system revealed its contribution to cell survival under unfavorable metabolic conditions. Indeed, G1-HIF plays a key role in the cell cycle-dependent expression of genes encoding metabolic regulators and the maintenance of mTOR activity under conditions of nutrient deprivation. Accordingly, transient elimination of G1-HIF led to a significant reduction in the concentration of key proteinogenic amino acids and carbohydrates. These data indicate that G1-HIF acts as a cell cycle-dependent surveillance factor that prevents the onset of starvation-induced apoptosis.

Phosphorylation of the PA subunit of influenza polymerase at Y393 prevents binding of the 5'-termini of RNA and polymerase function.

The influenza A virus (IAV) polymerase is a multifunctional machine that can adopt alternative configurations to perform transcription and replication of the viral RNA genome in a temporally ordered manner. Although the structure of polymerase is well understood, our knowledge of its regulation by phosphorylation is still incomplete. The heterotrimeric polymerase can be regulated by posttranslational modifications, but the endogenously occurring phosphorylations at the PA and PB2 subunits of the IAV polymerase have not been studied. Mutation of phosphosites in PB2 and PA subunits revealed that PA mutants resembling constitutive phosphorylation have a partial (S395) or complete (Y393) defect in the ability to synthesize mRNA and cRNA. As PA phosphorylation at Y393 prevents binding of the 5' promoter of the genomic RNA, recombinant viruses harboring such a mutation could not be rescued. These data show the functional relevance of PA phosphorylations to control the activity of viral polymerase during the influenza infectious cycle.

Release of STK24/25 suppression on MEKK3 signaling in endothelial cells confers cerebral cavernous malformation.

Loss-of-function mutations in cerebral cavernous malformation (CCM) genes and gain-of-function mutation in the MAP3K3 gene encoding MEKK3 cause CCM. Deficiency of CCM proteins leads to the activation of MEKK3-KLF2/4 signaling, but it is not clear how this occurs. Here, we demonstrate that deletion of the CCM3 interacting kinases STK24/25 in endothelial cells causes defects in vascular patterning during development as well as CCM lesion formation during postnatal life. While permanent deletion of STK24/25 in endothelial cells caused developmental defects of the vascular system, inducible postnatal deletion of STK24/25 impaired angiogenesis in the retina and brain. More importantly, deletion of STK24/25 in neonatal mice led to the development of severe CCM lesions. At the molecular level, a hybrid protein consisting of the STK kinase domain and the MEKK3 interacting domain of CCM2 rescued the vascular phenotype caused by the loss of ccm gene function in zebrafish. Our study suggests that CCM2/3 proteins act as adapters to allow recruitment of STK24/25 to limit the constitutive MEKK3 activity, thus contributing to vessel stability. Loss of STK24/25 causes MEKK3 activation, leading to CCM lesion formation.

Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex.

The influenza A virus (IAV)-encoded matrix protein 1 (M1) acts as a master regulator of virus replication and fulfills multiple structural and regulatory functions in different cell compartments. Therefore, the spatiotemporal regulation of M1 is achieved by different mechanisms, including its structural and pH-dependent flexibility, differential association with cellular factors, and posttranslational modifications. Here, we investigated the function of M1 phosphorylation at the evolutionarily conserved threonine 108 (T108) and found that its mutation to a nonphosphorylatable alanine prohibited virus replication. Absent T108, phosphorylation led to strongly increased self-association of M1 at the cell membrane and consequently prohibited its ability to enter the nucleus and to contribute to viral ribonucleoprotein nuclear export. M1 T108 phosphorylation also controls the binding affinity to the cellular STRIPAK (striatin-interacting phosphatases and kinases) complex, which contains different kinases and the phosphatase PP2A to shape phosphorylation-dependent signaling networks. IAV infection led to the redistribution of the STRIPAK scaffolding subunits STRN and STRN3 from the cell membrane to cytosolic and perinuclear clusters, where it colocalized with M1. Inactivation of the STRIPAK complex resulted in compromised M1 polymerization and IAV replication. IMPORTANCE Influenza viruses pose a major threat to human health and cause annual epidemics and occasional pandemics. Many virus-encoded proteins exert various functions in different subcellular compartments, as exemplified by the M1 protein, but the molecular mechanisms endowing the multiplicity of functions remain incompletely understood. Here, we report that phosphorylation of M1 at T108 is essential for virus replication and controls its propensity for self-association and nuclear localization. This phosphorylation also controls binding affinity of the M1 protein to the STRIPAK complex, which contributes to M1 polymerization and virus replication.

Testing the Effect of Histone Acetyltransferases on Local Chromatin Compaction.

Experiments determining the chromatin association of histone acetylases (HATs) and deacetylases (HDACs) at the genome-wide level provide precise maps of locus occupancy, but do not allow conclusions on the functional consequences of this locus-specific enrichment. Here we describe a protocol that allows tethering of HATs or HDACs to specific genomic loci upon fusion with a fluorescent protein and a DNA-binding protein such as the E. coli Lac repressor (LacI), which binds to genomically inserted lac operon sequences (lacO) via DNA/protein interactions. Integration of these lacO sequences into a genomic region of interest allows to monitor the functional consequences of HAT/HDAC targeting on chromatin (de)compaction, histone modification, and interaction with other proteins by quantitative light microscopy, as described here. As DNA-binding of LacI can be tightly controlled by the addition of galactose-derivatives, this method also allows to monitor the effects of locus-specific recruitment in a time-resolved manner.

Post-Transcriptional Control of mRNA Metabolism and Protein Secretion: The Third Level of Regulation within the NF-κB System.

The NF-κB system is a key transcriptional pathway that regulates innate and adaptive immunity because it triggers the activation and differentiation processes of lymphocytes and myeloid cells during immune responses. In most instances, binding to cytoplasmic inhibitory IκB proteins sequesters NF-κB into an inactive state, while a plethora of external triggers activate three complex signaling cascades that mediate the release and nuclear translocation of the NF-κB DNA-binding subunits. In addition to these cytosolic steps (level 1 of NF-κB regulation), NF-κB activity is also controlled in the nucleus by signaling events, cofactors and the chromatin environment to precisely determine chromatin recruitment and the specificity and timing of target gene transcription (level 2 of NF-κB regulation). Here, we discuss an additional layer of the NF-κB system that manifests in various steps of post-transcriptional gene expression and protein secretion. This less-studied regulatory level allows reduction of (transcriptional) noise and signal integration and endows time-shifted control of the secretion of inflammatory mediators. Detailed knowledge of these steps is important, as dysregulated post-transcriptional NF-κB signaling circuits are likely to foster chronic inflammation and contribute to the formation and maintenance of a tumor-promoting microenvironment.

Therapeutic induction of Bcl2-associated athanogene 3-mediated autophagy in idiopathic pulmonary fibrosis.

BACKGROUND: Exaggerated fibroblast proliferation is a well-known feature in idiopathic pulmonary fibrosis (IPF) which may be - in part - due to insufficient autophagy, a lysosome dependent cellular surveillance pathway. Bcl2-associated athanogene 3 (BAG3) is a pivotal co-chaperone of the autophagy pathway. Here, we studied whether therapeutic modulation of BAG3-mediated autophagy can rescue insufficient autophagy and impact IPF fibroblast proliferation. METHODS: Primary interstitial fibroblasts or precision cut lung slices (PCLS) of IPF lungs were treated with (1) the antifibrotic drug pirfenidone (Pirf), (2) the demethylating agent 5-azacytidine (Aza), (3) the BAG3 modulator cantharidin (Ctd). Autophagy flux was measured following pretreatment with the autophagy inhibitors or by GFP-RFP-LC3B transfection followed by drug treatments. Proliferation was measured by 5-bromo-2'-deoxyuridine assay. BAG3, filamin C (FLNC), proliferating-cell-nuclear-antigen (PCNA), collagen1A1 (COL1A1) and autophagy proteins were assessed by immunoblotting or immunofluorescence. Loss of function experiments were performed by siRNA mediated knockdown of BAG3. RESULTS: In comparison with healthy donors, increased BAG3 protein was observed in IPF lung homogenates and IPF fibroblasts. In addition, the substrate of BAG3-mediated autophagy, FLNC, was increased in IPF fibroblasts, implying insufficient activation of BAG3-dependent autophagy. Therapeutic modulation of this pathway using Aza and Ctd alone or in combination with the IPF therapy drug Pirf rescued the insufficient BAG3-mediated autophagy and decreased fibroblast proliferation. Such effects were observed upon therapeutic modulation of BAG3 but not upon knock down of BAG3 per se in IPF fibroblasts. Similarly, PCLS of IPF patients showed a significant decrease in collagen deposition in response to these drugs, either alone or in a more potent form in combination with Pirf. CONCLUSIONS: Our study reveals that repurposing drugs that modulate autophagy regulating proteins render therapeutic benefits in IPF. Fine tuning of this pathway may hence signify a promising therapeutic strategy to ameliorate antifibrotic properties and augment the efficacy of current IPF therapy.

Comparative kinase activity profiling of pathogenic influenza A viruses reveals new anti- and pro-viral protein kinases.

Constant evolution of influenza A viruses (IAVs) leads to the occurrence of new virus strains, which can cause epidemics and occasional pandemics. Here we compared two medically relevant IAVs, namely A/Hamburg/4/09 (H1N1pdm09) of the 2009 pandemic and the highly pathogenic avian IAV human isolate A/Thailand/1(KAN-1)/2004 (H5N1), for their ability to trigger intracellular phosphorylation patterns using a highly sensitive peptide-based kinase activity profiling approach. Virus-dependent tyrosine phosphorylations of substrate peptides largely overlap between the two viruses and are also strongly overrepresented in comparison to serine/threonine peptide phosphorylations. Both viruses trigger phosphorylations with distinct kinetics by overlapping and different kinases from which many form highly interconnected networks. As approximately half of the kinases forming a signalling hub have no known function for the IAV life cycle, we interrogated selected members of this group for their ability to interfere with IAV replication. These experiments revealed negative regulation of H1N1pdm09 and H5N1 replication by NUAK [novel (nua) kinase] kinases and by redundant ephrin A (EphA) receptor tyrosine kinases.

Thapsigargin: key to new host-directed coronavirus antivirals?

Despite the great success of vaccines that protect against RNA virus infections, and the development and clinical use of a limited number of RNA virus-specific drugs, there is still an urgent need for new classes of antiviral drugs against circulating or emerging RNA viruses. To date, it has proved difficult to efficiently suppress RNA virus replication by targeting host cell functions, and there are no approved drugs of this type. This opinion article discusses the recent discovery of a pronounced and sustained antiviral activity of the plant-derived natural compound thapsigargin against enveloped RNA viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), and influenza A virus. Based on its mechanisms of action, thapsigargin represents a new prototype of compounds with multimodal host-directed antiviral activity.

SIAH ubiquitin E3 ligases as modulators of inflammatory gene expression.

The functionally redundant ubiquitin E3 ligases SIAH1 and SIAH2 have been implicated in the regulation of metabolism and the hypoxic response, while their role in other signal-mediated processes such inflammatory gene expression remains to be defined. Here we have downregulated the expression of both SIAH proteins with specific siRNAs and investigated the functional consequences for IL-1α-induced gene expression. The knockdown of SIAH1/2 modulated the expression of approximately one third of IL-1α-regulated genes. These effects were not due to changes in the NF-κB and MAPK signaling pathways and rather employed further processes including those mediated by the coactivator p300. Most of the proteins encoded by SIAH1/2-regulated genes form a regulatory network of proinflammatory factors. Thus SIAH1/2 proteins function as variable rheostats that control the amplitude rather than the principal activation of the inflammatory gene response.

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival.

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin-Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.

Monitoring the Levels of Cellular NF-κB Activation States.

The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.

Multi-level inhibition of coronavirus replication by chemical ER stress.

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.