Critical Molecular Switch Research Articles

Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk.

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Abstract WP300: Ifi27l2a is a Novel Target for Dampening Microglial Inflammation Following Stroke

Microglia play a critical role in initiating and spreading inflammation and are involved in the chronic recovery phase following stroke. Microglia express multiple interferon-stimulated genes (ISGs) in response to inflammatory cues, including Ifi27l2a . However, the specific roles of individual ISGs, such as Ifi27l2a , have not been well-studied in the context of stroke. We tested the hypothesis that induction of Ifi27l2a in microglia contributes to the pro-inflammatory response following stroke. We used Ifi27l2a heterozygote (Het) mice to determine the effect of partial reduction of Ifi27l2a in primary microglia and in an experimental stroke model. Primary microglia isolated from WT and Het brains were stimulated with LPS to investigate the role of Ifi27l2a in microglial activation in vitro . Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in young mice (WT vs Het, male, 3 months old) to determine whether deletion of Ifi27l2a reduces microglial activation and improves functional outcome following stroke. We found that LPS-induced expression of Il1b was markedly reduced in Het microglia compared to WT microglia (n=6, p<0.01). Moreover, other inflammation marker genes ( Il1a and Tnfa ) were significantly reduced in Het microglia compared to WT microglia in LPS-stimulated conditions (n=6, p<0.05). The levels of inducible forms of Mmp3 and Mmp9 , which are responsible for blood-brain barrier (BBB) dysfunction, were significantly reduced in Het microglia (n=3, p<0.01). However, Mmp2 expression was not altered by deletion of Ifi27l2a. These findings suggest that Ifi27l2a is involved in the process of BBB dysfunction and microglial inflammation. Lastly, gliosis (microgliosis and astrogliosis) was significantly reduced at 14 days post-stroke in the thalamus of Het brains, a known site of secondary injury in pdMCAO model (n=6, p<0.05). Foot fault tests showed improved functional outcome in Het mice (n=4-6, p<0.05). Ifi27l2a appears to be a critical molecular switch for inflammatory mechanisms in the post-stroke brain. These findings suggest that interventions targeting Ifi27l2a regulation (even partial inhibition) could reduce harmful inflammation and promote improved functional outcome following stroke.

Demethylation of EHMT1/GLP Protein Reprograms Its Transcriptional Activity and Promotes Prostate Cancer Progression.

In this study, we demonstrate that EHMT1 and EHMT2 proteins drive prostate cancer development by transcriptionally activating multiple oncogenic pathways. Mechanistically, the chromatin binding of EHMT1 is significantly expanded through demethylation of both lysine 450 and 451 residues, which can serve as a critical molecular switch to induce oncogenic transcriptional reprogramming in prostate cancer cells.

Understanding the role of the LRRK2 tail in the crosstalk between kinase and the N-terminal domains.

LRRK2 is a multi-domain protein encompassing biology's two most critical molecular switches; a kinase and a GTPase. Mutations in LRRK2 are one of the key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to exploring the physiological role of LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is a common feature in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and can serve as a multi-functional protein: protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and are using peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB region with a combination of Gaussian MD simulations and peptide arrays. This Ct-Motif contains multiple phosphorylation sites including two auto-phosphorylation sites (Thr2083 and Thr2524), a 14-3-3 binding site (Thr2524) and a putative PKA phosphorylation site. These two sites together form a part of a dynamic “CAP” that regulates the N-lobe of the kinase domain. We also explore how the cellular localization of LRRK2RCKW and its docking onto microtubules is altered by mutations in the Ct helix, if at all. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

OA07.02 TAZ Regulates SCLC Phenotypic Transition and Metastasis

Small cell lung cancer (SCLC) remains as the most lethal form of lung cancer with very poor prognosis. High metastatic capability and strong plasticity is the hallmark of SCLC. However, the exact cell subpopulation responsible for SCLC metastasis and critical regulators in contribution to SCLC plasticity still remain largely unclear. Growing evidence has revealed the importance of epigenetic reprograming during cancer malignant progression. Mammalian SWI/SNF complex BAF is an adenosine triphosphate (ATP)-dependent multi-component machinery, essential for remodeling chromatin architecture. Smarca4 (Brg1), the core ATPase component of BAF complex is preferentially required for the progression of SCLC with MAX (Myc-associated factor) inactivation. Also, it still remains elusive about the exact function of Brg1 and SWI/SNF complex in SCLC metastasis. Using the well-established Rb1L/L/Trp53 L/L (RP) mouse model, we here identify the NCAMhiCD44lo/- subpopulation derived from RP model as SCLC metastasizing cells (SMC). Moreover, we find that the SMC is progressively transitioned from the NCAMloCD44hi cells (Non-SCLC metastasizing cells, Non-SMC) during malignant progression of SCLC. Through combined analysis of ATAC-seq and RNA-seq data of SMC vs. non-SMC, we observe a dramatic alteration in chromatin accessibility between these two subpopulations, and TAZ, the core transcription cofactor of the Hippo pathway, is epigenetically silenced by the SWI/SNF complex during SCLC malignant progression. Knockout of Brg1 in RP mouse promotes TAZ expression and abrogates the phenotype transition from Non-SMC to SMC and almost completely blocks SCLC metastasis. Allograft assay further confirms TAZ as the critical molecular switch during the phenotypic transition and SCLC metastasis. In this study, we identify the NCAMhiCD44lo/- subpopulation derived from RP model as SMC subpopulation, which is progressively transitioned from the Non-SMC during SCLC malignant progression. Through combined analysis of ATAC-seq and RNA-seq data, we observe a dramatic alteration in chromatin accessibility between these two subpopulations, which points to the alteration of SWI/SNF complex activation. We further find that genetic disruption of the SWI/SNF chromatin-remodeling complex through Brg1 knockout abrogates the phenotype transition from Non-SMC to SMC and blocks SCLC metastasis. Mechanistically, we find that TAZ, the core transcription cofactor of the Hippo pathway, is epigenetically silenced by the SWI/SNF complex. Down-regulation of TAZ promotes the transition from Non-SMC to SMC and facilitates SCLC metastasis. Conversely, ectopic TAZ expression or TAZ activation by digitoxin treatment reversibly promotes the transition from SMC to Non-SMC and thus alleviates SCLC metastasis. Importantly, the treatment of digitoxin, a clinically used drug for heart failure treatment, promotes TAZ activation and inhibits SCLC phenotypic transition and metastasis. Through the immunostaining analyses of 101 Chinese SCLC specimens, we further find that low TAZ expression significantly correlates with SMC features and poor patient prognosis. Collectively, these data uncover an important subpopulation in contribution to SCLC metastasis and highlights TAZ as the critical molecular switch in this process. Moreover, TAZ activation by digitoxin treatment provides an effective avenue to control SCLC phenotypic transition and metastasis, providing a potential therapeutic avenue for the metastatic SCLC management in clinic.

Unlocking the Recovery Potential: JMJD3 Inhibition-Mediated SAPK/JNK Signaling Inactivation Supports Endogenous Oligodendrocyte-Lineage Commitment Post Mammalian Spinal Cord Injury

Spinal cord injury (SCI) induced catastrophic neurological disability is often incurable at present. The injury triggered immediately oligodendrocytes loss and overwhelming demyelination are regarded as an insurmountable barrier to SCI recovery. To date, effective strategy to promote the endogenous oligodendrocytes replacement post SCI remains elusive. Epigenetic modifications are emerging as critical molecular switches of gene expression in CNS. However, the epigenetic mechanisms underlying oligodendrogenesis post SCI yet to be discovered. In this study, we report that H3K27me3 demethylase JMJD3 exists as a pivotal epigenetic regulator which manipulates the endogenous oligodendrogenesis post SCI. We found that JMJD3 inhibition promotes the oligodendrocyte linage commitment of neural stem/progenitor cells (NPCs) in vitro and in vivo. Moreover, we demonstrated that JMJD3 inhibition mediated SAPK/JNK signaling inactivation is functionally necessary for endogenous oligodendrocyte-lineage commitment post SCI. Our results also suggested that JMJD3 is downstream of SAPK/JNK pathway, and capable of translates SCI induced SAPK/JNK signaling into epigenetic codes readable by spinal cord endogenous NPCs. Taken together, our findings provide novel evidence of JMJD3 mediated oligodendrocyte-lineage commitment orchestration post SCI, which would be a potential epigenetic approach to induce the mature mammalian endogenous recovery.

Identification and characterization of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) involved in regulating extracellular ATP-mediated innate immune responses in Japanese flounder (Paralichthys olivaceus)

Extracellular adenosine triphosphate (eATP), released following inflammatory stimulation or infection, is a potent signaling molecule in activating innate immune responses in fish. However, the regulation of eATP-mediated innate immunity in fish remains unknown. Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a critical molecular switch for controlling the ATP levels in the extracellular space. CD39 plays a key role in regulating eATP-activated innate immune responses through the phosphohydrolysis of pro-inflammatory eATP to inactive AMP. Here, we identified and characterized a CD39 homolog (namely, poCD39) in the Japanese flounder Paralichthys olivaceus and analyzed its regulatory role in eATP-mediated innate immunity. Real-time quantitative PCR analysis revealed that poCD39 is ubiquitously present in all tested normal tissues with dominant expression in enriched Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments demonstrated that poCD39 expression was upregulated by inflammatory stimulation and Edwardsiella tarda infection. Biochemical and immunofluorescence analysis revealed that poCD39 is a functional glycosylated membrane protein for the hydrolysis of eATP. Inhibition of poCD939 activity with the ecto-NTPDase inhibitor ARL 67156 resulted in increased IL-1beta gene expression and ROS production in Japanese flounder HKMs. In contrast, overexpression of poCD39 in Japanese flounder FG-9307 cells reduced eATP-induced pro-inflammatory cytokine IL-1beta gene expression. Finally, poCD39 expression was significantly induced by eATP stimulation in the HKMs, suggesting that eATP may provide a feedback mechanism for transcriptional regulation of fish CD39. Taken together, we identified and characterized a functional fish CD39 protein involved in regulating eATP-mediated innate immune responses in fish.

MicroRNA-142-mediated repression of phosphodiesterase 3B critically regulates peripheral immune tolerance.

Tregs play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). This function is dependent on maintenance of a high intracellular cAMP concentration. A number of microRNAs are implicated in the maintenance of Tregs. In this study, we demonstrate that peripheral immune tolerance is critically dependent on posttranscriptional repression of the cAMP-hydrolyzing enzyme phosphodiesterase-3b (Pde3b) by microRNA-142-5p (miR-142-5p). In this manner, miR-142-5p acts as an immunometabolic regulator of intracellular cAMP, controlling Treg suppressive function. Mir142 was associated with a super enhancer bound by the Treg lineage–determining transcription factor forkhead box P3 (FOXP3), and Treg-specific deletion of miR-142 in mice (TregΔ142) resulted in spontaneous, lethal, multisystem autoimmunity, despite preserved numbers of phenotypically normal Tregs. Pharmacological inhibition and genetic ablation of PDE3B prevented autoimmune disease and reversed the impaired suppressive function of Tregs in TregΔ142 animals. These findings reveal a critical molecular switch, specifying Treg function through the modulation of a highly conserved, cell-intrinsic metabolic pathway. Modulation of this pathway has direct relevance to the pathogenesis and treatment of autoimmunity and cancer.

Loss of LRRC25 accelerates pathological cardiac hypertrophy through promoting fibrosis and inflammation regulated by TGF-β1

Despite advances in therapeutic strategies, heart failure-associated mortality rates remain high. Thus, understanding the pathophysiological molecular mechanisms involved in the remodeling process is essential for developing new and effective therapies. LRRs are present various prokaryotic and eukaryotic proteins and important for the innate immune system via regulating protein-protein interactions. LRRC25 is a member of leucine-rich repeat (LRR)-containing protein family. LRRC25 has been shown to negatively modulate nuclear factor κB (NF-κB) activation, a crucial factor related to cardiac hypertrophy. Our aim was to explore the effects of LRRC25 on cardiac hypertrophy. In the present study, LRRC25 levels were decreased in human and mouse hypertrophied hearts. LRRC25 knockout exacerbated cardiac hypertrophy responding to pressure overloading or angiotensin II (Ang II) stimulation. Deletion of LRRC25 accelerated cardiac dysfunction and fibrosis in mice subjected to aortic banding (AB). LRRC25 ablation induced a strong increase in the transcription of both hypertrophy (ANP, BNP, and β-MHC) and fibrosis associated molecules (col1, col3a1, α-SMA and fibronectin). In addition, the expression of transforming growth factor-β1 (TGF-β1), and its down-streaming signals of phosphorylated Smad2/3, was markedly induced by LRRC25 deficiency. LRRC25-knockout mice showed a significantly enhanced inflammation in response to AB surgery by promoting the activation of NF-κB signaling pathway. In mouse cardiomyocytes, LRRC25 deficiency markedly elevated TGF-β1 and NF-κB activation stimulated by Ang II. Treatment with a combination of TGF-β1 or NF-κB inhibitor abolished the effects of LRRC25-knockout on the promotion of cardiac hypertrophy in vitro. Together, our study identified LRRC25 as a critical molecular switch whose down-regulation resulted in cardiac hypertrophy in a TGF-β1- and NF-κB-dependent manner.

The Low-Threshold Calcium Channel Cav3.2 Mediates Burst Firing of Mature Dentate Granule Cells.

Mature granule cells are poorly excitable neurons that were recently shown to fire action potentials, preferentially in bursts. It is believed that the particularly pronounced short-term facilitation of mossy fiber synapses makes granule cell bursting a very effective means of properly transferring information to CA3. However, the mechanism underlying the unique bursting behavior of mature granule cells is currently unknown. Here, we show that Cav3.2 T-type channels at the axon initial segment are responsible for burst firing of mature granule cells in rats and mice. Accordingly, Cav3.2 knockout mice fire tonic spikes and exhibit impaired bursting, synaptic plasticity and dentate-to-CA3 communication. The data show that Cav3.2 channels are strong modulators of bursting and can be considered a critical molecular switch that enables effective information transfer from mature granule cells to the CA3 pyramids.

Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148

AbstractSrc family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)– and hemi-ITAM–containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)–containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring.

OsLAP6/OsPKS1, an orthologue of Arabidopsis PKSA/LAP6, is critical for proper pollen exine formation

BackgroundMale fertility is crucial for rice yield, and the improvement of rice yield requires hybrid production that depends on male sterile lines. Although recent studies have revealed several important genes in male reproductive development, our understanding of the mechanisms of rice pollen development remains unclear.ResultsWe identified a rice mutant oslap6 with complete male sterile phenotype caused by defects in pollen exine formation. By using the MutMap method, we found that a single nucleotide polymorphism (SNP) variation located in the second exon of OsLAP6/OsPKS1 was responsible for the mutant phenotype. OsLAP6/OsPKS1 is an orthologous gene of Arabidopsis PKSA/LAP6, which functions in sporopollenin metabolism. Several other loss-of-function mutants of OsLAP6/OsPKS1 generated by the CRISPR/Cas9 genomic editing tool also exhibited the same phenotype of male sterility. Our cellular analysis suggested that OsLAP6/OsPKS1 might regulate pollen exine formation by affecting bacula elongation. Expression examination indicated that OsLAP6/OsPKS1 is specifically expressed in tapetum, and its product is localized to the endoplasmic reticulum (ER). Protein sequence analysis indicated that OsLAP6/OsPKS1 is conserved in land plants.ConclusionsOsLAP6/OsPKS1 is a critical molecular switch for rice male fertility by participating in a conserved sporopollenin precursor biosynthetic pathway in land plants. Manipulation of OsLAP6/OsPKS1 has potential for application in hybrid rice breeding.

Global profiling of Rbm24 bound RNAs uncovers a multi-tasking RNA binding protein.

RNA binding proteins serve as critical molecular switches in a multitude of post-transcriptional regulatory processes. In the heart and muscles, the tissue specific RNA binding protein, Rbm24, is known to play important developmental roles via driving different post-transcriptional processes. Nonetheless, the currently identified molecular targets and regulatory pathways seem inadequate to completely explain the observed developmental effects upon Rbm24 knockdown/knockout. Here, by performing RNA Immunoprecipitation and coupling it to microarrays (RIP-Chip), we have generated an atlas of the mRNA binding repertoire of Rbm24. Further functional evaluation of its targets led to the elucidation of novel roles for Rbm24 in post-transcriptional processing, besides its already known roles in regulation of mRNA stability and alternative splicing. Interestingly, Rbm24 is found to cause the destabilization of Chrm2 via binding to an element in the coding region. In addition, Rbm24 is also found to have an uncharacterized role in driving the generation of isoforms with alternative transcriptional start sites. We have, for the first time, demonstrated that Rbm24 is a multi-tasking RNA binding protein capable of regulating its bound targets via a range of mechanisms.

YAP/TAZ and Hedgehog Coordinate Growth and Patterning in Gastrointestinal Mesenchyme

YAP/TAZ are the major mediators of mammalian Hippo signaling; however, their precise function in the gastrointestinal tract remains poorly understood. Here we dissect the distinct roles of YAP/TAZ inendodermal epithelium and mesenchyme and findthat, although dispensable for gastrointestinal epithelial development and homeostasis, YAP/TAZ function as the critical molecular switch to coordinate growth and patterning in gut mesenchyme. Our genetic analyses reveal that Lats1/2 kinases suppress expansion of the primitive mesenchymal progenitors, where YAP activation also prevents induction of the smooth muscle lineage through transcriptional repression of Myocardin. During later development, zone-restricted downregulation of YAP/TAZ provides the positional cue and allows smooth muscle cell differentiation induced by Hedgehog signaling. Taken together, our studies identify the mesenchymal requirementof YAP/TAZ in the gastrointestinal tract and highlight the functional interplays between Hippo and Hedgehog signaling underlying temporal and spatial control of tissue growth and specification in developing gut.

Abstract 5010: Activation of the notch signaling pathway confers a tumor-suppressive phenotype on melanoma-associated fibroblasts

Abstract Objectives. The tumor microenvironment (TME) is an emerging therapeutic target for cancer treatment. Cancer-associated fibroblasts (CAF) play a crucial role in cancer progression. We aim to target TME by altering intracellular signaling which determines the biological function of CAF. We have recently showed that the Notch signaling pathway likely functions as a molecular switch in controlling the tumor regulatory role of CAF in animal models and experimentally created “CAF”. Here, we investigated the status of Notch signaling in human melanoma-associated fibroblasts (MAF) versus their normal counterparts and tested whether manipulation of the Notch pathway activity in MAF alter their tumor-regulating function. Methods. We examined levels of Hes1, a canonical Notch target, in MAF of human malignant melanoma at different stages (I-IV) and fibroblasts in either adjacent or non-adjacent normal skin tissues using tissue microarray. MAF were isolated from human metastatic melanoma tissues. Notch pathway RT2-PCRArray and immunoblot were used to assess Notch pathway activity in MAF versus normal human dermal fibroblasts. Activation of Notch signaling pathway in MAF was achieved by lentiviral vector encoding active form of Notch1 (NIC). The effect of Notch1-engineered MAF on melanoma growth was tested by in vitro co-cultures and in a mouse co-xenograft model (n=6/group). Tumor angiogenesis was analyzed by immunochemistry. Results. MAF expressed decreased levels of Hes1 compared with adjacent skin fibroblasts. Isolated MAF also exhibited lower Notch activity than normal human dermal fibroblasts. Notch1-engineered MAF significantly inhibited melanoma cell growth in vitro (p<0.01) and suppressed melanoma growth and tumor angiogenesis in mice (p<0.05). Conclusions. Notch pathway activity is down-regulated in MAF. Increase of Notch pathway activity confers MAF with inhibition to melanoma growth and tumor angiogenesis. Our study demonstrates that Notch signaling is a critical molecular switch in determining the tumor regulatory role of MAF and provides potential targets for cancer therapeutic interventions on the TME. Citation Format: Hongwei Shao, Mecker G. Moller, Long Cai, Leiming Zhang, Zhao-Jun Liu. Activation of the notch signaling pathway confers a tumor-suppressive phenotype on melanoma-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2017-5010

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy

ABSTRACTNRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.

ALMS1 acts as a critical molecular switch that controls Insulin-stimulated glucose transport in adipocytes

ALMS1 acts as a critical molecular switch that controls Insulin-stimulated glucose transport in adipocytes

Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses.

An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.

Integration of Cardiac Troponin I Phosphorylations to Modulate Function

The troponin complex is a critical molecular switch involved in transducing the calcium activating signal into contraction. Troponin I (TnI), the inhibitory subunit, is phosphorylated as a key mechanism to alter the calcium regulation of contraction. The canonical phosphorylation of TnI at serine-23/24 (Ser-23/24) is a significant modulator of this regulation, and may be fine-tuned by other TnI phosphorylation events. A novel TnI phosphorylation at tyrosine 26 (Tyr-26) was identified in the human heart and shown to decrease in heart failure. We have recently demonstrated that this novel TnI Tyr-26 phosphorylation decreases thin filament calcium sensitivity and accelerates calcium dissociation by itself, identical to the functional effects of Ser-23/24. We further demonstrated the integration of these two modifications causes an additive effect on calcium dissociation while maintaining a decreased calcium sensitivity. These data stress the importance of Tyr-26 function, especially in the presence of Ser-23/24. These data further highlight how the integration of TnI phosphorylation exhibits differential effects when compared to studying these events in isolation. Importantly, the integration of Ser-23/24 and Tyr-26 phosphorylation may not be limited to functional effects alone, but may manifest in signaling as well. Considering the tyrosine kinase recognition sequence (EEXY) is similar to that surrounding TnI Tyr-26 when Ser-23/24 are occupied with phosphate(S(p)S(p)NY), we hypothesized that Ser-23/24 phosphorylation would exhibit crosstalk, altering phosphorylation rate of Tyr-26. To assess this hypothesis, we developed an antibody against pTnI Tyr-26 as a tool to measure changes in Tyr-26 phosphorylation. We validated the specificity and purity of this antibody and are currently elucidating signaling integration by assessing the ability of Ser-23/24 phosphorylation to regulate phosphorylation of Tyr-26 by tyrosine kinases. The functional and signaling integration of TnI phosphorylation may therefore be a significant mechanism to modulate Ser-23/24 phosphorylation function.