Novel TDP-43 stress reporter models to accelerate drug development in ALS.

The development of therapies to combat neurodegenerative diseases is widely recognized as a research priority. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of reporters of cytoprotective pathways (e.

CK2 phosphorylation of CMTR1 promotes RNA cap formation and influenza virus infection.

The RNA cap methyltransferase CMTR1 methylates the first transcribed nucleotide of RNA polymerase II transcripts, impacting gene expression mechanisms, including during innate immune responses. Using mass spectrometry, we identify a multiply phosphorylated region of CMTR1 (phospho-patch [P-Patch]), which is a substrate for the kinase CK2 (casein kinase II). CMTR1 phosphorylation alters intramolecular interactions, increases recruitment to RNA polymerase II, and promotes RNA cap methylation.

Potential of stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies.

Chemical risk assessment ensures protection from the toxic effects of drugs and manmade chemicals. To comply with regulatory guidance, studies in complex organisms are required, as well as mechanistic studies to establish the relevance of any toxicities observed to man. Although toxicity models are improving, studies remain central to this process.

Defining the in vivo mechanism of air pollutant toxicity using murine stress response biomarkers.

Air pollution can cause a wide range of serious human diseases. For the informed instigation of interventions which prevent these outcomes there is an urgent need to develop robust in vivo biomarkers which provide insights into mechanisms of toxicity and relate pollutants to specific adverse outcomes. We exemplify for a first time the application of in vivo stress response reporters in establishing mechanisms of air pollution toxicity and the application of this knowledge in epidemiological studies.

In vivo stress reporters as early biomarkers of the cellular changes associated with progeria.

Age-related diseases account for a high proportion of the total global burden of disease. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo stress reporter systems as surrogate early biomarkers of the degenerative disease progression.

Application of the in vivo oxidative stress reporter Hmox1 as mechanistic biomarker of arsenic toxicity.

Inorganic arsenic (iAs) is a naturally occurring metalloid present in drinking water and polluted air exposing millions of people globally. Epidemiological studies have linked iAs exposure to the development of numerous diseases including cognitive impairment, cardiovascular failure and cancer. Despite intense research, an effective therapy for chronic arsenicosis has yet to be developed.

DHX15 regulates CMTR1-dependent gene expression and cell proliferation.

CMTR1 contributes to mRNA cap formation by methylating the first transcribed nucleotide ribose at the O-2 position. mRNA cap O-2 methylation has roles in mRNA stabilisation and translation, and self-RNA tolerance in innate immunity. We report that CMTR1 is recruited to serine-5-phosphorylated RNA Pol II C-terminal domain, early in transcription.

Regulation and function of CMTR1-dependent mRNA cap methylation.

mRNA is modified co-transcriptionally at the 5' end by the addition of an inverted guanosine cap structure which can be methylated at several positions. The mRNA cap recruits proteins involved in gene expression and identifies the transcript as being cellular or 'self' in the innate immune response. Methylation of the first transcribed nucleotide on the ribose 2'-O position is a prevalent cap modification which has roles in splicing, translation and provides protection against the innate immune response.

CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription.

The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine.

G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization.

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain.

The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.

Discovery of potent and selective covalent inhibitors of JNK.

The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.

Alternative ERK5 regulation by phosphorylation during the cell cycle.

ERK5 is a member of the mitogen-activated protein kinase (MAPK) family that, after stimulation, is activated selectively by dual phosphorylation in the TEY motif by MAPK kinase 5 (MEK5). ERK5 plays an important role in regulating cell proliferation, survival, differentiation and stress response. Moreover, it is involved in G2/M progression and timely mitotic entry.

ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis.

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear.

p38gamma regulates interaction of nuclear PSF and RNA with the tumour-suppressor hDlg in response to osmotic shock.

Activation of p38γ modulates the integrity of the complex formed by the human discs large protein (hDlg) with cytoskeletal proteins, which is important for cell adaptation to changes in environmental osmolarity. Here we report that, in response to hyperosmotic stress, p38γ also regulates formation of complexes between hDlg and the nuclear protein polypyrimidine tract-binding protein-associated-splicing factor (PSF). Following osmotic shock, p38γ in the cell nucleus increases its association with nuclear hDlg, thereby causing dissociation of hDlg-PSF complexes.

Differential activation of p38MAPK isoforms by MKK6 and MKK3.

All four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family (p38alpha, p38beta, p38gamma and p38delta) are activated by dual phosphorylation in the TGY motif in the activation loop. This phosphorylation is mediated by three kinases, MKK3, MKK6 and MKK4, at least in vitro. The role of these MKK in the activation of p38alpha has been demonstrated in studies using fibroblasts that lack MKK3 and/or MKK6.

Proteolysis of the tumour suppressor hDlg in response to osmotic stress is mediated by caspases and independent of phosphorylation.

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is a component of the p38gamma MAP kinase pathway, which is important for the adaptation of mammalian cells to changes in environmental osmolarity. Here we report a strong decrease in the levels of hDlg protein in the human epithelial cell line HeLa when exposed to osmotic shock.

Modulation of sarcoplasmic reticulum Ca(2+)-ATPase by chronic and acute exposure to peroxynitrite.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SERCA), an integral membrane protein, becomes irreversibly inactivated in vitro by the addition of a single bolus of peroxynitrite with a K(0.5) of 200-300 microm, and this results in a large decrease of the ATP-dependent Ca2+ gradient across the sarcoplasmic reticulum (SR) membranes. The inactivation of SERCA is raised by treatment of SR vesicles with repetitive micromolar pulses of peroxynitrite.