Biology subjects

Gaestel, M.

Publications and source records attributed to Gaestel, M..

14 recordsLinked to original sources

bioRxiv2025

MK2/p38/p53 negatively regulate basal IL-1β and non-canonical NF-κB signaling

Interleukin (IL)-1{beta} is a pro-inflammatory cytokine implicated in sterile inflammation and tumor development. Investigating the role of MAPKAP kinase 2 (MK2) in IL-1{beta} processing, we found that Il1b mRNA and IL-1{beta} protein levels were elevated in resting MK2-knockout (KO) macrophages and in the serum of MK2/3-double-KO mice. This was linked to activation of the non-canonical NF-{kappa}B pathway in the absence of MK2 or its activator, p38. Rescue by MK2, its kinase-inactive mutant MK2K79R, or p38 suppressed this pathway and reduced Il1b expression. We also observed decreased basal protein levels of tumor suppressor p53 in MK2-or p38-deficient cells. Mechanistically, p53 interacts with mitochondrial caspase-3, promoting cleavage of RelB, thereby inhibiting non-canonical NF-{kappa}B signaling and subsequent Il1b and TP53 expression. These findings explain elevated basal IL-1{beta} levels in MK2-KO macrophages and uncover a new autoregulatory mechanism of TP53 expression. Additionally, they reveal a new mechanism that contributes to the long-discussed link between cancer and inflammation, wherein the tumor suppressor p53 inhibits cytokine production in parallel.

biochemistry↗

bioRxiv2025

Septin7 is essential in early hematopoiesis, but redundant at later stages

The unique cytoskeletal protein Septin7 is generally considered to be required for cytokinesis in yeast and mammals. Whole body genetic ablation of Septin7 in mice is embryonic lethal. Septin7-deficient fibroblasts and HeLa cells are defective in cytokinesis and undergo obligate multinucleation. Surprisingly, lymphocyte- and myeloid-specific deletion of Septin7 in mice did not result in any detectable abnormalities in blood lineage development, suggesting that Septin7 is dispensable for steady-state hematopoiesis. In contrast, Septin7-deficient hematopoietic stem cells failed to engraft and establish donor chimerism following transplantation, indicating that Septin7 is essential for hematopoietic stem cell function under stress conditions. To reconcile these contradictory findings, we analyzed the effects of Septin7 deletion in hematopoietic cells from mice with either pan-hematopoietic or lymphoid lineage-specific Septin7 deletion. Our results demonstrate that Cre-induced deletion of the floxed Septin7 allele is inefficient during the early stages of hematopoiesis, suggesting strong selection pressure against Septin7 deficiency at this stage. In contrast, deletion of Septin7 at the common lymphoid progenitor stage, as well as in Hoxb8-immortalized hematopoietic progenitors, is efficient and does not result in any noticeable defects in cell division. Taken together, our findings indicate that Septin7 is essential during early hematopoiesis but becomes dispensable at later stages.

biochemistry↗

bioRxiv2023

MK2 deficiency decreases mortality during the inflammatory phase after myocardial infarction in mice

BackgroundAltering the onset, intensity, or duration of inflammation can impact the recovering hearts structure and function following myocardial infarction (MI). Substrates of MAP kinase-activated protein kinase 2 (MK2) include proteins that regulate the stability of AU-rich transcripts, including those of several pro-inflammatory cytokines. This study was to determine if MK2-deficiency impaired the inflammatory phase of post-MI wound repair. Methods and ResultsMyocardial infarctions were induced by permanent ligation of the left anterior descending coronary artery in 12-week-old male MK2+/+ and MK2-/- littermate mice. Five days post-MI, survival was 100% in MI-MK2-/- (n = 20) and 79% in MI-MK2+/+ mice (n = 29; Mandel-Cox test: P < 0.05). Area at risk and infarct size were similar. Echocardiographic imaging revealed that both systolic and diastolic LV diameters were greater in MI-MK2+/+ than MI-MK2-/- mice. MK2-deficiency did not affect the increase in wall motion score index. Infiltration of neutrophils or monocytes did not differ significantly. Cytokine and chemokine transcripts were quantified in infarcted and non-infarcted LV tissue using qPCR arrays (QIAGEN). Three days post-MI, Ifna2 was increased and Il16 was decreased in infarcted tissue from MK2-/- hearts, compared with infarcted MK2+/+ tissue, whereas in the non-infarcted MK2-/- myocardium Il27 increased and Tnfsf11, Ccl3, and Il1rn were decreased. Five days post-MI, Ctf16 and Il10 increased in infarcted MK2-/- tissue whereas in the non-infarcted MK2-/- myocardium Ccl9, Nodal, and Xcl2 increased and Il15 decreased. ConclusionsThe findings of this study suggest MK2-deficiency is an advantage during the inflammatory phase of cardiac wound repair post-MI. Clinical PerspectiveWhat is new? -The effects of MAP kinase-activated protein kinase 2 (MK2) deficiency on survival, cardiac structure and function, and the inflammatory phase of wound healing following myocardial infarction were assessed using a constitutive, pan MK2-null mouse model. -MK2-deficiency reduced mortality but did not alter area at risk or infarct size post-myocardial infarction. Inflammatory cell infiltration was also unaffected. -MK2-deficiency altered the abundance of several cytokines (increased, decreased) in infarcted and non-infarcted myocardium post-MI. What are the clinical implications? -The initial phase of wound repair post-MI involves inflammation. -The risk of damage to the myocardium and mortality may be reduced by inhibition of MK2 activity during the inflammatory phase of wound healing post-MI.

physiology↗

bioRxiv2023

5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFkappaB signaling

Receptor-interacting protein kinases (RIPK) -1 and -3 are master regulators of cell fate decisions in response to diverse stimuli and are subjected to multiple checkpoint controls. Earlier studies have established the presence of distinct IKK1/2 and p38/MK2-dependent checkpoints which suppress RIPK1 activation by directly phosphorylating it at different residues. In the present study, we investigated TNF-induced death in MAPK-activated protein kinase 2 (MK2)-deficient cells and show that MK2-deficiency or inactivation predominantly results in necroptotic cell death, even in the absence of caspase inhibition. While MK2-deficient cells can be rescued from necroptosis by RIPK1 inhibitors, RIPK3 inhibition seems to revert the process triggering apoptosis. To understand the mechanism of this necroptosis switch, we screened a 149-compound kinase inhibitor library for compounds which preferentially sensitize MK2-deficient MEFs to TNF-induced cell death. The most potent inhibitor identified was 5-Iodotubericidin, an adenosine analogue acting as adenosine kinase and protein kinase inhibitor. 5-ITu also potentiated LPS-induced necroptosis when combined with MK2 inhibition in RAW264.7 macrophages. Further mechanistic studies revealed that 5-Iodotubericidin induces RIPK1-dependent necroptosis in the absence of MK2 activity by suppressing IKK signaling. The identification of this role for the multitarget kinase inhibitor 5-ITu in TNF-, LPS- and chemotherapeutics-induced necroptosis will have potential implications in RIPK1-targeted therapies.

cell biology↗

bioRxiv2023

Enforced dimerization of CD45 by the adenovirus E3/49K protein inhibits T cell receptor signaling

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals, but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D adenoviruses. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45 causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both the secreted sec49K and the membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer.

microbiology↗

bioRxiv2022

On the therapeutic potential of MAPK4 in triple-negative breast cancer

ERK3/MAPK6 (MAPK6 gene) along with its paralog ERK4/MAPK4 (MAPK4 gene) define a distinct subfamily of atypical mitogen-activated protein kinases (MAPKs)1. Much remains to be learned about the substrates and biological functions of these signaling enzymes. Interestingly, recent work has suggested that ERK4 promotes prostate cancer progression via the non-canonical activation of AKT/mTOR signaling2,3. In their recent study, Wang et al.4 report that ERK4 is expressed in a subset of triple-negative breast cancer (TNBC) cell lines and that this expression is critical for AKT activation and for sustaining TNBC cell proliferation in vitro and tumor growth in mice. They also show that depletion of ERK4 sensitizes TNBC cells to phosphatidylinositol-3-kinase (PI3K) inhibitors. They conclude that ERK4 is a promising therapeutic target for TNBC and has potential for combination therapy with PI3K inhibitors. Here, we raise concerns about the cellular models and experimental approaches used in this study, which compromises the conclusions on the oncogenic role of ERK4 in TNBC.

cancer biology↗

bioRxiv2022

PRMT5-mediated regulatory arginine methylation of RIPK3

The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell-survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context dependent manner. Using a mass spectrometry-based screen to find new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3 (R486 in human, R415 in mouse) was identified as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5-mediated RIPK3 methylation to act as an efficient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling.

cell biology↗

bioRxiv2022

Pharmacological activation of the mitochondrial stress protease OMA1 reveals a therapeutic liability in Diffuse Large B-Cell Lymphoma

Constitutive activation of the ATF4-mediated integrated stress response (ATF4-ISR) is common in cancer and buffers the metabolic challenges imposed by rapid proliferation. However, hyperactivation of the ISR can induce apoptosis. Here we demonstrate that novel pyrazolo-thiazole derivates activate the mitochondrial protease OMA1 which subsequently induces apoptosis in diffuse large B-cell lymphoma (DLBCL) cells. Apoptosis is dependent on the OMA1 mediated cleavage of DELE1 which leads to activation of HRI and induction of the ATF4 ISR. Screening in 406 cancer cell lines identified an inverse correlation between sensitivity to OMA1 activators and expression of the mitochondrial protein FAM210B. Ectopic overexpression of FAM210B specifically blocks OMA1 activation and apoptosis induction by pyrazolo-thiazole activators in DLBCL. OMA1 activators, including the preclinical candidate BTM-3566, selectively killed ABC, GCB, and double-hit DLBCL lines and induced complete tumor regression across a panel of DLBCL patient-derived xenografts. SignificanceHere we describe a novel class of small molecules that activate the mitochondrial protease OMA1 and induce therapeutic responses in DLBCL preclinical models in vitro and in vivo. OMA1 activation drives apoptosis through ATF4-ISR, an orthogonal mechanism to current therapies.

cancer biology↗

bioRxiv2021

ERK3-MK5 signaling regulates myogenic differentiation and muscle regeneration by promoting FoxO3 degradation

The physiological functions and downstream effectors of the atypical mitogen-activated protein kinase ERK3 remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6KD/KD) exhibit a reduced post-natal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs post-natal skeletal muscle growth and adult muscle regeneration after injury. Loss of MK5 phenocopies the muscle phenotypes of Mapk6KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control post-natal myogenic differentiation.

cell biology↗

bioRxiv2021

A Cdc42-Borg4-Septin 7 axis regulates HSCs polarity and function

Aging of hematopoietic stem cells (HSCs) is caused by an elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal septin network. Here we show that Cdc42 interacts with borg4, which in turn interacts with septin 7 to regulate the polar distribution of Cdc42, borg4 and septin 7 within HSCs. Genetic deletion of either borg4 or septin 7 in HSCs resulted in a reduced frequency of HSCs polar for Cdc42 or borg4 or septin 7 and a reduced engraftment potential and decreased lymphoid-primed multipotent progenitors (LMPPs) frequency in the bone marrow. In aggregation our data identify a Cdc42-borg4-septin 7 axis to be essential for maintenance of polarity within HSCs and for HSC function and provide rationale for further investigating the role of borgs and septins for the regulation of compartmentalization within stem cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/435817v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@168dd5org.highwire.dtl.DTLVardef@b9b4a3org.highwire.dtl.DTLVardef@26e05org.highwire.dtl.DTLVardef@1851fcf_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

MAP kinase-activated protein kinase-5 (MK5) plays an important role in cardiac fibroblast function. Although p38 MAPK and atypical MAPKs and ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. Here we examined the expression, subcellular distribution, and regulation of MK5 in cardiac ventricular myofibroblasts and myocytes. The copy numbers for MK5 and ERK4 mRNA were comparable in myocytes and myofibroblasts, whereas that of ERK3 was much higher in myofibroblasts. Interestingly, MK5 and ERK3 immunoreactivity was detected in myofibroblasts but not myocytes whereas ERK4 immunoreactivity was detected in myocytes: treating in myocytes with a proteasome inhibitor or hypertrophic agonists failed to rescue MK5 immunoreactivity. In myofibroblasts, MK5 and ERK3 immunoreactivity was predominantly nuclear and cytosolic, respectively. In serum-starved cardiac myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGF{beta}, or H2O2 and this was prevented by inhibition of p38/{beta}. Phos-tag SDS-PAGE revealed multiple slower migrating bands of MK5 immunoreactivity, indicating phosphorylation of MK5 at multiple sites. Phos-tag PAGE also revealed MK5 phosphorylation was increased with fibroblast activation and in hearts exposed to a chronic increase in afterload. MK5 and ERK3 co-immunoprecipitated and proximity ligation assays revealed ERK3 and MK5 in close proximity in myofibroblast cytoplasmic compartment. Furthermore, p38/{beta} inhibition decreased the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. Finally, deleting MK5 did not reduce the abundance of ERK3 immunoreactivity. These observations suggest that p38 and/or p38{beta} are the primary mediators of T182-MK5 phosphorylation and hence MK5 activation in cardiac myofibroblasts.

biochemistry↗

bioRxiv2020

MK2-deficient mice are bradycardic and display delayed hypertrophic remodelling in response to a chronic increase in afterload

MAP kinase-activated protein kinase-2 (MK2) is protein serine/threonine kinase activated by p38/{beta}. Herein we examined the cardiac phenotype of pan MK2-null (MK2-/-) mice. Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P = 0.580). At 12-weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, ageing was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3 were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+-sensitivity to mitochondrial permeability transition pore (mPTP) opening. Finally, the pressure overload-induced increase in heart weight/tibia length and decrease in systolic function were attenuated in MK2-/- mice two weeks, but not eight weeks, after constriction of the transverse aorta. Collectively, these results implicate MK2 in (i) autonomic regulation of heart rate, (ii) cardiac mitochondrial function, and (iii) the early stages of myocardial remodeling in response to chronic pressure overload. Key points summaryThe cardiac characterization of pan MK2-null mice showed: O_LIaltered autonomic regulation of heart rate C_LIO_LIincreased expression of key metabolic genes C_LIO_LIdecreased Ca2+-sensitivity for MPTP opening C_LIO_LIdelayed hypertrophic remodeling in response to increased afterload C_LI

physiology↗

bioRxiv2018

Germline deletion reveals a non-essential role of the atypical MAPK6/ERK3

MAPK6/ERK3 is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary disfunction and by defects in function, activation and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon3 flanked by LoxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing first evidence for the existence of the ERK3/MK5 signaling complex in vivo. In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile, do not display pulmonary hypoplasia, acute respiratory failure, abnormal T cell development, reduction of thymocyte numbers or altered T cells selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality, lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin-resistance-cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal suggesting redundant functions of both protein kinases.

cell biology↗

bioRxiv2018

Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

Shaping of the proteome by alternative translation is an important mechanism of post-transcriptional gene regulation. It can lead to the expression of multiple protein isoforms originating from the same mRNA. Here we show that a novel, abundant and long isoform of the stress/p38MAPK-activated kinase MK2, a key regulator of transcription, migration, death signaling and post-transcriptional gene regulation, is constitutively translated from an alternative CUG translation initiation start site located in the 5'UTR of its mRNA. GC-rich sequences and putative G-quadruplex structures influence the usage of that codon as a translation initiation start site and the RNA helicase eIF4A1 is needed to ensure alternative isoform translation. We recapitulated the usage of the alternative start codon and determined the molecular properties of the short and a long MK2 isoforms. Phenotypically, only the short isoform phosphorylated Hsp27, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling by quantitative mass-spectrometry revealed short isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains additional putative phosphorylation sites in its unique N-terminus. In sum, our data reveal a longer and previously non-described isoform of MK2 with distinct physiological properties originating from alternative translation.

molecular biology↗