Podcasts about Phosphorylation

Drs. Jason Crowell and Subhojit Roy discuss evidence that suggests phosphorylated α-synuclein might actually have a role in normal brain cell function. Show Reference: https://www.cell.com/neuron/fulltext/S0896-6273(23)00894-2?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0896627323008942%3Fshowall%3Dtrue 

Dr. Jason Crowell talks with Dr. Subhojit Roy about a platform for investigating the role of serine-129 in synucleinopathies, with implications for drug development. Read the related article in the Neuron. Disclosures can be found at Neurology.org.

References Nature . 2019 Nov;575(7782):361-365. Adv Biol Regul. 2017 May:64:39-48 J Biol Chem. 2013 Apr 5; 288(14): 9933–9945 Redox Biology.2017. Volume 11, April. Pages 622-630 Biochim Biophys Acta. 2011 Jun;1811(6):377-85 Dr Guerra: lipid lectures archives Schubert, F. 1816. Symphony #5 https://youtu.be/OHkot1TmvZU?si=uYtQfxUegl3KrkRK Newman, L Darby K. 1954. Marilyn Monroe. The River of No Return https://youtu.be/dLzeHkEQe9g?si=nXMd20gstc3vVdgl --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

References Biochim Biophys Acta. 2011 Jun;1811(6):377-85 Am J Physiol Lung Cell Mol Physiol 2001. 281: L98–L107. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 1997. Volume 1348, Issues 1–2, 4 Pages 79-90. Am J Physiol Lung Cell Mol Physiol 2001. Volume 281. Issue 1July Pages L108-L118 JBC 1991. Volume 266, Issue 36, 25 December. Pages 24503-24508 Progress in Lipid Research. 2015. Volume 59, ages 147-171 Nature 2019. volume 575, pages 361–365 --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

References Mol Biol Cell. 2001 Sep; 12(9): 2711–2720. Small GTPases. 2014; 5: e28579. Cells. 2021 Jul 20;10(7):1831. J Investig Med. 2023 Feb;71(2):113-123 --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

In this week's episode, we discuss the impact of silent cerebral infarction in patients with immune mediated thrombotic thrombocytopenic purpura (or iTTP) in clinical remission, how the survival of leukemia stem cells is highly dependent on oxidative phosphorylation in the mitochondria, and targeting iron import as a potential therapeutic approach in aggressive natural killer cell leukemia (or ANKL).

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550331v1?rss=1 Authors: Yucel, B. P., Al Momany, E., Evans, A. J., Seager, R., Wilkinson, K., Henley, J. M. Abstract: Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550521v1?rss=1 Authors: Dou, D., Aiken, J., Holzbaur, E. L. F. Abstract: Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1B{beta} complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550435v1?rss=1 Authors: Bradley, R. A., Wolff, I. D., Cohen, P. E., Gray, S. Abstract: During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of homologous chromosomes during the first meiotic division. The mechanism by which DSBs become selected to repair as crossovers is unknown, although the crossover positioning and levels in each cell indicate it is a highly regulated process. One of the proteins that localises to crossover sites is the serine/threonine cyclin-dependent kinase CDK2. Regulation of CDK2 occurs via phosphorylation at tyrosine 15 (Y15) and threonine 160 (T160) inhibiting and activating the kinase, respectively. In this study we use a combination of immunofluorescence staining on spread spermatocytes and fixed testis sections, and STA-PUT gravitational sedimentation to isolate cells at different developmental stages to further investigate the temporal phospho regulation of CDK2 during prophase I. Western blotting reveals differential levels of the two CDK2 isoforms (CDK233kDa and CDK239kDa) throughout prophase I, with inhibitory phosphorylation of CDK2 at Y15 occurring early in prophase I, localising to telomeres and diminishing as cells enter pachynema. Conversely, the activatory phosphorylation on T160 occurs later, specifically the CDK233kDa isoform, and T160 signal is detected in spermatogonia and pachytene spermatocytes, where it co-localises with the Class I crossover protein MLH3. Taken together, our data reveals intricate control of CDK2 both with regards to levels of the two CDK2 isoforms, and differential regulation via inhibitory and activatory phosphorylation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.11.548513v1?rss=1 Authors: Han, Y., Li, M., Zhao, B., Wang, H., Liu, Y., Liu, Z., Xu, J., Yang, R. Abstract: Microtubule based vesicle transport is an essential way for protein delivery. Selective transport of vesicles to soma-dendritic area or to axon is critical for maintaining neuron polarity. Although neuronal axon is full filled with plus-end-out microtubules, vesicles containing dendritic protein, like TfR labeled vesicles, barely been transported to the axon. How does neuron maintain the transport of TfR labeled vesicles dendritically? Using live cell imaging and loss of function assay, we found that KIF13A is a major transporter for TfR vesicles at soma-dendritic area. Over-expression of KIF13A drives dendritic TfR vesicles to axon. We further found that MARK2 binds to and phosphorylates KIF13A at 14-3-3 binding motif inhibiting its kinesin activity at the axon initial segment (AIS), which is critical for maintaining TfR vesicle at the soma-dendritic area. Overall, our results suggested a novel kinesin-based mechanism for dendritic-selective transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547506v1?rss=1 Authors: Kehlenbach, R. H., James, C., Möller, U., König, S., Urlaub, H. Abstract: ELYS is a nucleoporin that localizes to the nuclear side of the nuclear envelope in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate the formation of novel nuclear pore complexes. Here we describe the interaction of ELYS with the membrane protein VAPB. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which is shown to mediate interaction with the MSP (major sperm protein)-domain of VAPB. Phosphorylation-dependent binding of VAPB to ELYS is demonstrated by peptide binding assays and co-immunoprecipitation experiments. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB resulted in prolonged mitosis and slow progression from meta- to anaphase and also to chromosome segregation defects. Together, our results suggest an active role of VAPB in recruiting membrane fragments to chromatin and in the biogenesis of a novel nuclear envelope during mitosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547549v1?rss=1 Authors: Tzeng, C. P., Whitwam, T., Boxer, L. D., Li, E., Silberfeld, A., Trowbridge, S., Mei, K., Lin, C., Shamah, R., Griffith, E. C., Renthal, W., Chen, C., Greenberg, M. E. Abstract: Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the capacity of the brain to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

References Guerra Biochemistry lecture archives Nat Metab. 2022 Sep;4(9):1202-1213 Mol Cell. 2008 May 23; 30(4): 403–414 J Clin Invest. 2016 Nov 1; 126(11):4361–4371. Front Immunol. 2021; 12: 746151. --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message

References Int J Mol Sci. 2020 Jun; 21(11): 4098. Authentic Biochemistry lectures :ImmunoEpigenetics 56-58 --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539436v1?rss=1 Authors: Smythe, E., Alshahrani, F., Zhu, Z., Ferreira, F., Maib, H., Pruzina, S., Robinson, D., Murray, D. H., McComb, A. Abstract: Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537129v1?rss=1 Authors: Hardt, R., Dehghani, A., Schoor, C., Goedderz, M., Cengiz Winter, N., Ahmadi, S., Sharma, R., Schork, K., Eisenacher, M., Gieselmann, V., Winter, D. Abstract: Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify double cortin like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.09.536178v1?rss=1 Authors: Wang, S., Sivadasan Bindu, D., Tan, C. X., Sakers, K., Takano, T., Rodriguez Salazar, M. P., Dimond, K., Soderling, S. H., La Spada, A. R., Eroglu, C. Abstract: Astrocytes tightly control neuronal connectivity and function in the brain through direct contact with synapses. These glial cells become reactive during disease pathogenesis including Parkinson's disease (PD). However, it remains unknown if astrocyte dysfunction is an initiating factor of PD pathogenesis and whether astrocytes can be targeted to stop or reverse the synaptic dysfunction seen in PD. Using in vitro and in vivo methods, we found that the PD-linked gene Lrrk2 controls astrocyte morphology via regulating the phosphorylation of ERM proteins (Ezrin, Radixin, and Moesin), a structural component of the perisynaptic astrocyte processes. ERM phosphorylation is robustly elevated both in mice and humans carrying the LRRK2 G2019S Parkinsonism mutation. Importantly, the reduction of the ERM phosphorylation, specifically in the LRRK2 G2019S in adult astrocytes, is sufficient to restore excitatory synapse number and function deficits in the LRRK2 G2019S knock-in mouse cortex. These results show a role for Lrrk2 in controlling astrocyte morphogenesis and synaptogenic function and reveal that early astrocyte dysfunction in PD could be causal to disruptions in cortical excitatory synaptic connectivity. The astrocytic dysfunction can be corrected by dampening ERM phosphorylation, pinpointing astrocytes as critical cellular targets for PD therapeutics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535705v1?rss=1 Authors: Yang, L., Parajuli, N., Wu, P., Liu, J., Wang, X. Abstract: Background: A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that cAMP-dependent protein kinase (PKA) activates the 26S proteasome by phosphorylating Ser14 of RPN6 (pS14-RPN6), but this discovery and its physiological significance remain to be established in vivo. Methods: Male and female mice with Ser14 of Rpn6 mutated to Ala (S14A) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system (UPS) functioning was evaluated with the GFPdgn reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G). Results: PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type (WT) but not S14A embryonic fibroblasts (MEFs), adult cardiomyocytes (AMCMs), and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and AMCMs than in WT counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with non-transgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than WT neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates and of aberrant CryAB protein aggregates, less reactivation of fetal genes and cardiac hypertrophy, and delays in cardiac malfunction. Conclusions: This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifies a new therapeutic target to reduce cardiac proteotoxicity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534397v1?rss=1 Authors: Smolen, K. A., Papke, C. M., Swingle, M. R., Musiyenko, A., Li, C., Camp, A. D., Honkanen, R. E., Kettenbach, A. N. Abstract: Variants in the phosphoprotein phosphatase-2 regulatory protein-5D gene (PPP2R5D) cause the clinical phenotype of Jordan's Syndrome (PPP2R5D-related disorder), which includes intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ~40% of reported cases. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G greater than A) in the PPP2R5D allele in a heterozygous manner in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of wild-type, E198K, and E420K cell lines and find unique and shared changes between variants and wild-type cells in kinase- and phosphatase-controlled signaling cascades. As shared signaling alterations, we observed ribosomal protein S6 (RPS6) hyperphosphorylation, indicative of increased ribosomal protein S6-kinase activity. Rapamycin treatment suppressed RPS6 phosphorylation in both, suggesting activation of mTORC1. Intriguingly, our data suggest AKT-dependent (E420K) and -independent (E198K) activation of mTORC1. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, treatment with rapamycin or a p70S6K inhibitor warrants further investigation as potential therapeutic strategies for patients. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.15.532613v1?rss=1 Authors: Kharod, S. C., Hwang, D., Castillo, P. E., Yoon, Y. J. Abstract: Fragile X messenger ribonucleoprotein (FMRP) is an RNA-binding protein that can form granules within neurons. Although FMRP-containing Fragile X granules have been extensively characterized, visualizing changes in granule structure in relation to function has been difficult. We used high resolution imaging to detect discrete differences in puncta size, intensity and contrast in our mouse FMRP and serine 499 (S499) phospho-mutant reporters. Tracking analyses of FMRP particles showed that phospho-mutant granules traveled at rates consistent with active transport within dendrites, while GFP-FMRP exhibited a wide range of speeds owing to its ability to oligomerize when overexpressed. Taken together, our approach allows us to evaluate FMRP granules and elucidate the central role of FMRP phosphorylation and dephosphorylation in granule clustering and transport. It had been recently suggested that FMRP granules could be phase separated structures that may function as hubs for local protein synthesis. To test this idea, we performed fluorescence recovery after photobleaching (FRAP) on individual FMRP granules. Intriguingly, we observed only a partial recovery that occurred over minutes time-scale and was dependent on translation. Moreover, the phospho-mutants revealed differences in recovery kinetics reflecting the labile (S499A) or preferential (S499D) nature of their interaction with granules containing stalled ribosomes. Our high resolution approach can reveal how the phosphorylation-state of FMRP can modulate the clustering of FMRP granules and thereby regulate local protein synthesis at the level of individual granules. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.13.532494v1?rss=1 Authors: Dong, Y., Wang, Y., Qi, T., Zhang, X., Shen, L., Ma, J., Pang, Z., McClatchy, D. B., Lal, N., Wang, K., Xie, Y., Polli, F., Maximov, A., Augustine, V., Cline, H. T., Yates, J. R., Ye, L. Abstract: For decades, the expression of immediate early genes (IEGs) such as c-fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

REferences Dr Guerra's graduate biochemistry lectures J Biol Chem. 2013 Mar 8; 288(10): 7137–7146 --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.28.530230v1?rss=1 Authors: Harris, R. J., Heer, M., Levasseur, M. D., Cartwright, T. N., Weston, B., Mitchell, J. L., Coxhead, J. M., Gaughan, L., Prendergast, L., Rico, D., Higgins, J. M. G. Abstract: Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where particular combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognise trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me3 during mitosis. We find that H3K4me3 hinders adjacent H3T3ph deposition in cells, and that the PHD domain of TAF3 can bind H3K4me3 in mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.27.530364v1?rss=1 Authors: Berkane, R., Ho-Xuan, H., Glogger, M., Sanz-Martinez, P., Cano-Franco, S., Juretschke, T., Gonzales Cardenas, A., Glaesner, T., Beli, P., Husnjak, K., Doetsch, V., Grumati, P., Heilemann, M., Stolz, A. Abstract: Selective autophagy of the ER (ERphagy) is an important regulator of ER remodeling and critical to maintain cellular homeostasis upon environmental changes. ERphagy receptors link the ER with autophagic membrane thus regulating ERphagy flux. We recently showed that members of the FAM134 family play overlapping and distinct roles during stress-induced ERphagy. Yet the mechanisms on how they are activated remain largely unknown. In this study we analyzed mTOR-mediated dynamic phosphorylation of FAM134 as a trigger of FAM134-driven ERphagy. An unbiased screen of kinase inhibitors revealed CK2 to be essential for FAM134B- and FAM134C-driven ERphagy upon mTOR inhibition. Identified dynamic phosphorylation sites on FAM134C in cells were fitting with predicted CK2 targeting sites, indicating a direct regulatory role of CK2 in FAM134-driven ERphagy. Using super-resolution microscopy, we showed that activity of CK2 is essential for the formation of high-density clusters of FAM134B and FAM134C. Consistently, FAM134B and FAM134C proteins carrying point mutations of selected Serin residues, within their reticulon homology domain, are unable to form high-density clusters. In addition, we provide evidence that the ubiquitination machinery is required for ERphagy and that FAM134B and FAM134C clustering is activated by phospho-dependent ubiquitination. Treatment with CK2 inhibitor SGC-CK2-1 prevents Torin1-induced ERphagy flux as well as ubiquitination of FAM134 proteins and consistently, treatment with E1 inhibitor suppresses Torin1-induced ERphagy flux. Therefore, we propose CK2 dependent phosphorylation of ERphagy receptors precedes ubiquitin-dependent ERphagy flux activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.19.528206v1?rss=1 Authors: Chapelet, G., Beguin, N., Castellano, B., Grit, I., Oullier, T., Neunlist, M., Blottiere, H., Rolli-Derkinderen, M., Le Drean, G., Derkinderen, P. Abstract: There is mounting evidence to suggest that the gut-brain axis is involved in the development of Parkinson's disease (PD). In this regard, the enteroendocrine cells (EEC), which faces the gut lumen and are connected with both enteric neurons and glial cells have received growing attention. The recent observation showing that these cells express alpha-synuclein, a presynaptic neuronal protein genetically and neuropathologically linked to PD came to reinforce the assumption that EEC might be a key component of the neural circuit between the gut lumen and the brain for the bottom-up propagation of PD pathology. Besides alpha-synuclein, tau is another key protein involved in neurodegeneration and converging evidences indicate that there is an interplay between these two proteins at both molecular and pathological levels. There are no existing studies on tau in EEC and therefore we set out to examine the isoform profile and phosphorylation state of tau in these cells. Methods Surgical specimens of human colon from control subjects were analyzed by immunohistochemistry using a panel of anti-tau antibodies together with chromogranin A and Glucagon-like peptide-1 (two EEC markers) antibodies. To investigate tau phosphorylation and expression further, two EEC lines, namely GLUTag and NCI-H716 were analyzed by western blot after dephosphorylation with pan-tau and tau isoform specific antibodies. Eventually, GLUTag were treated with propionate and butyrate, two short chain fatty acids known to sense EEC, and analyzed at different time points by western blot with an antibody specific for tau phosphorylated at Thr205. Results We found that tau is expressed and phosphorylated in EEC in adult human colon and that both EEC lines mainly express two tau isoforms that are phosphorylated under basal condition. Both propionate and butyrate regulated tau phosphorylation state by decreasing its phosphorylation at Thr205. Conclusion and inference Our study is the first to characterize tau in human EEC and in EEC lines. As a whole, our findings provide a basis to unravel the functions of tau in EEC and to further investigate the possibility of pathological changes in tauopathies and synucleinopathies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.19.529131v1?rss=1 Authors: Isei, M., Girardi, P., Rodwell-Bullock, J., nehrke, k., Johnson, G. V. Abstract: Phosphorylation of tau at sites associated with Alzheimer's disease (AD) likely plays a role in the disease progression. Mitochondrial impairment, correlating with increased presence of phosphorylated tau, has been identified as a contributing factor to neurodegenerative processes in AD. However, how tau phosphorylated at specific sites impacts mitochondrial function has not been fully defined. We examined how AD-relevant phosphomimetics of tau impact selected aspects of mitochondrial biology. To mimic phosphorylation at AD-associated sites, the Ser/Thr sites in wild-type GFP tagged-tau (T4) were converted to glutamic acid (E) to make pseudophosphorylated GFP tagged-Ser-396/404 (2EC) and GFP tagged-Thr-231/Ser-235 (2EM) constructs. These constructs were expressed in neuronal HT22 cells and their impact on specific mitochondrial functions and responses to stressors were measured. Phosphomimetic tau altered mitochondrial distribution. Specifically, mitochondria accumulated in the soma of cells expressing either 2EC or 2EM, and neurite-like extensions in 2EC cells were shorter. Additionally, ATP levels were reduced in both 2EC and 2EM expressing cells, and ROS production increased in 2EC cells during oxidation of succinate when compared to T4 expressing cells. Thapsigargin reduced mitochondrial membrane potential ({Psi}m) and increased ROS production in both 2EC and 2EM cells relative to T4 cells, with no significant difference in the effects of rotenone. These results show that tau phosphorylation at specific AD-relevant epitopes negatively affects mitochondria, with the extent of dysfunction and stress response varying according to the sites of phosphorylation. Altogether, these findings extend our understanding of potential mechanisms whereby phosphorylated tau promotes mitochondria dysfunction in tauopathies, including AD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

References Cell Death & Differentiation 2020.27: 3374–3385 Cell Death & Differentiation 2019. 26: 1501–1515 Cancers 2020, 12(8), 2137. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.15.528749v1?rss=1 Authors: Maxson, M. E., Huynh, K., Grinstein, S. Abstract: While it has been known for decades that luminal acidification is required for normal traffic along the endocytic pathway, the precise underlying mechanism(s) remain unknown. We found that dissipation of the endomembrane pH gradient resulted in acute formation of large Rab5- or Rab7-positive vacuoles. Vacuole formation was associated with and required hyperactivation of the Rabs, which was attributable to impaired GTPase activity, despite normal recruitment of cognate GAPs. Surprisingly, LRRK2 -a kinase linked to Parkinsons disease-was recruited to endomembranes and markedly activated upon dissipation of luminal acidification. LRRK2 phosphorylated Rab GTPases, rendering them insensitive to deactivation. Importantly, genetic deletion of LRRK2 prevented the {Delta}pH-induced vacuolation, implying that the kinase is required to modulate vesicular traffic. We propose that by dictating the state of activation of LRRK2 and in turn that of Rab GTPases, the development of a progressive luminal acidification serves as a timing device to control endocytic maturation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.13.528057v1?rss=1 Authors: Jaeckel, E., Arias-Hervert, E. R., Perez-Medina, A., Herrera, Y., Schulz, S. S., Birdsong, W. T. Abstract: Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well-understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) and anterior cingulate cortex (ACC) via activity at -opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in both the DMS and ACC. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses, MThal-ACC synapses, and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation), but decreased subsequent morphine inhibition of transmission at MThal-ACC synapses (morphine tolerance) in a sex-specific manner; these adaptations were present in male but not female mice. Additionally, these adaptations were not observed in knockin mice expressing phosphorylation-deficient MORs, suggesting a role of MOR phosphorylation in mediating both facilitation and tolerance to morphine within this circuit. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry and sex. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.22.525045v1?rss=1 Authors: Harmanda, B., Kaya, O., Waide, X., Qureshi, M. H., Nesvizhskii, A., Mitchison, T., Ozlu, N. Abstract: Keratins are the most diverse family of intermediate filaments and are expressed in most epithelial tissues and malignancies. They form highly stable polymers that need to be cut through during cytokinesis. Previous work suggested a role of phosphorylation, but keratin regulation during cell division is not understood in detail. Depletion of Keratin 8 in an epithelial cancer cell line (HeLa) caused chromosome segregation and cytokinesis defects. Aurora B kinase localization to midzones and cleavage furrows was reduced in Keratin 8 knockouts, suggesting that Keratin 8 helps scaffold Aurora B during cytokinesis. We mapped eleven Aurora B kinase sites in Keratin 8 that were associated with cell division. Keratin 8 S34 phosphorylation occurred specifically at the cleavage furrow and persisted at the midzone until the end of cytokinesis. Inhibition of Aurora B kinase or non-phosphorylatable Keratin 8 mutant prevented the disassembly of keratin bundles at the cleavage furrow, which blocked furrow ingression. Our data reveal a functional inter-dependency between Keratin 8 and Aurora B kinase in epithelial cells. Keratin 8 promotes the translocation of Aurora B to the midzone. Aurora B kinase then generates a local zone of Keratin phosphorylation which promotes keratin filament disassembly and allows the cleavage furrow to cut through the keratin network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.11.523603v1?rss=1 Authors: Ziołkowska, M., Borczyk, M., Cały, A., Nalberczak-Skora, M., Nowacka, A., Sliwinska, M. A., Łukasiewicz, K., Skonieczna, E., Tomaszewski, K. F., Wojtowicz, T., Wlodarczyk, J., Bernas, T., Salamian, A., Radwanska, K. Abstract: The updating of contextual memories is essential for survival in a changing environment. Accumulating data indicate that the dorsal CA1 area (dCA1) contributes to this process. However, the cellular and molecular mechanisms of contextual fear memory updating remain poorly understood. Postsynaptic density protein 95 (PSD-95) regulates the structure and function of glutamatergic synapses. Here, using dCA1-targeted genetic manipulations in vivo, combined with ex vivo 3D electron microscopy and electrophysiology, we identify a novel, synaptic mechanism that is induced during attenuation of contextual fear memories and involves phosphorylation of PSD-95 at Serine 73 in dCA1. Our data provide the proof that PSD-95-dependent synaptic plasticity in dCA1 is required for updating of contextual fear memory. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.11.523290v1?rss=1 Authors: Mazzarino, R. C., Perez-Corredor, P. A., Vanderleest, T. E., Vacano, G. N., Sanchez, J., Villalba-Moreno, N. D., Krasemann, S., Mendivil Perez, M. A., Aguillon, D., Jimenez-Del-Rio, M., Baena, A., Sepulveda-Falla, D., Lopera-Restrepo, F., Quiroz-Gaviria, Y., Arboleda-Velasquez, J. F. Abstract: Alzheimer's disease (AD) is the most common cause of dementia among older adults. APOE3 Christchurch (R136S, APOE3Ch) variant homozygosity was reported in an individual with extreme resistance to autosomal dominant AD due to the PSEN1 E280A mutation. This subject had a delayed clinical age at onset and resistance to tauopathy and neurodegeneration despite extremely high amyloid plaque burden. We established induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and from a non-protected kindred control (with PSEN1 E280A and APOE3/3). We used CRISPR/Cas9 gene editing to successfully remove the APOE3Ch to wild type in iPS cells from the protected case and to introduce the APOE3Ch as homozygote in iPS cells from the non-protected case to examine causality. We found significant reduction of tau phosphorylation (pTau 202/205 and pTau396) in cerebral organoids with the APOE3Ch variant, consistent with the strikingly reduced tau pathology found in the resistant case. We identified Cadherin and Wnt pathways as signaling mechanisms regulated by the APOE3Ch variant through single cell RNA sequencing in cerebral organoids. We also identified elevated {beta}-catenin protein, a regulator of tau phosphorylation, as a candidate mediator of APOE3Ch resistance to tauopathy. Our findings show that APOE3Ch is necessary and sufficient to confer resistance to tauopathy in an experimental ex-vivo model establishing a foundation for the development of novel, protected case-inspired therapeutics for tauopathies, including Alzheimer's. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

This podcast covers the effect of phophorylstion/dephosphorylation by protein kinases and phosphatases on enzyme activity. This biochemistry content may be useful to premedical and medical students. Similar content is available at: MEDBIOCHEM.ORG --- Send in a voice message: https://anchor.fm/a-j-ghalayini/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.05.522745v1?rss=1 Authors: Jimenez, A. J., Bousquet, H., Bardin, S., Perez, F., Goud, B., Miserey, S. Abstract: RAB GTPases are key regulators of membrane trafficking in eukaryotic cells. In addition to their role in interphase, several RAB proteins, including Golgi-associated RAB6, have mitotic functions. The aim of this study was to investigate how the interphasic and mitotic functions of RAB6 could be regulated. Since phosphorylation is a key regulatory process in mitosis, we looked for specific mitotic phosphorylation of RAB6 using a phospho-proteomic approach. We found that RAB6 is phosphorylated at position S52 by the mitotic kinase Polo-like kinase 1 (Plk1) in mitosis. Phosphorylated RAB6 localizes at the spindle poles from prophase to anaphase. In metaphase, we observed RAB6A-positive structures containing Mad1 and Mad2 moving along the mitotic spindle via the dynein-dynactin complex. We provide evidence that phosphorylation impairs RAB6A binding to some of its known partners, including p150Glued and Bicaudal-D2. In addition, the overexpression of RAB6A phospho-mutants lead to mitosis and cytokinesis defects. Our results suggest that a cycle of RAB6 phosphorylation/dephosphorylation is required for cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.24.521856v1?rss=1 Authors: Bulgari, D., Cavolo, S. L., Schmidt, B. F., Buchan, K., Bruchez, M. P., Deitcher, D. L., Levitan, E. Abstract: Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins such as the 70 kDa protease TPA, which contributes to long-term potentiation and synaptic growth. Rather than using full collapse exocytosis that is common in endocrine cells, DCVs at a native intact synapse, the Drosophila neuromuscular junction, release their contents via fusion pores formed by kiss and run exocytosis. Here fluorogen activating protein (FAP) imaging reveals the limited permeability range of synaptic DCV fusion pores and then shows that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+ independent full fusions require PKA-R2, a PKA phosphorylation site on the fusion clamp protein complexin and the acute presynaptic function of Rugose/Neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent signaling triggers the opening of dilating fusion pores to release large DCV cargo proteins that cannot pass through fusion pores that normally dominate Ca2+-dependent synaptic protein release. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

References Neural Plasticity. 2019; 2019: 1648736. Molecular Pharmacology May 2017, 91 (5) 451-463. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.22.521485v1?rss=1 Authors: Parra-Rivas, L. A., Madhivanan, K., Wang, L., Boyer, N. P., Prakashchand, D. D., Aulston, B. D., Pizzo, D. P., Branes-Guerrero, K., Tang, Y., Das, U., Scott, D. A., Rangamani, P., Roy, S. Abstract: Phosphorylation of -synuclein at the Serine-129 site (-syn Ser129P) is an established pathologic hallmark of synucleinopathies, and also a therapeutic target. In physiologic states, only a small fraction of total -syn is phosphorylated at this site, and consequently, almost all studies to date have focused on putative pathologic roles of this post-translational modification. We noticed that unlike native (total) -syn that is widely expressed throughout the brain, the overall pattern of -syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser-129 site blocked the ability of -syn to attenuate activity-dependent synaptic vesicle (SV) recycling; widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments -syn Ser-129P, and this phosphorylation is required for -syn binding to VAMP2 and synapsin - two functional binding-partners that are necessary for -syn function. AlphaFold2-driven modeling suggests a scenario where Ser129P induces conformational changes in the C-terminus that stabilizes this region and facilitates protein-protein interactions. Our experiments indicate that the pathology-associated Ser129P is an unexpected physiologic trigger of -syn function, which has broad implications for pathophysiology and drug-development. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.21.521356v1?rss=1 Authors: Komatsu, M., Ikeda, R., Noshiro, D., Morishita, H., Takada, S., Kageyama, S., Fujioka, Y., Funakoshi, T., Komatsu-Hirota, S., Arai, R., Ryzhii, E., Abe, M., Koga, T., Nakao, M., Sakimura, K., Horii, A., Waguri, S., Ichimura, Y., Noda, N. N. Abstract: NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of phosphorylation remain unclear. Herein, we identify ULK1 as a kinase responsible for phosphorylation of p62. ULK1 co-localizes with p62 bodies, and directly interacts with p62. This phosphorylation allows KEAP1 to be retained within p62 bodies, activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351 corresponding to human Ser349 is replaced by Glu. These mice, but not phosphodefective p62S351A/S351A mice, exhibit NRF2 hyperactivation and growth retardation, the latter caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis. p62S351E/+ mice are a phenocopy of systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insight into the role of phase separation in this process. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.19.521109v1?rss=1 Authors: Kumar, S., Stainer, A., Dubrulle, J., Simpkins, C., Cooper, J. Abstract: Integrins link the cytoskeleton to the extracellular matrix for cell movement. Integrin ligation stimulates tyrosine phosphorylation of integrin-associated proteins but the role of phosphorylation signaling in regulating integrin-cytoskeletal linkages and assembly of focal adhesions is unclear. Using spreading or migrating epithelial cells, we provide evidence that phosphorylated Cas (p130Cas, BCAR1), its binding partner, Crk, and inactive focal adhesion kinase (FAK) cluster together with inactive integrins at the cell periphery at sites that develop into focal adhesions containing F-actin, active integrins, active FAK and mechanosensing proteins such as vinculin and talin. Cas, Crk, Src family kinases (SFK) and Rac1 are required for focal adhesion formation and cell spreading, while vinculin is not needed for Cas clustering. Furthermore, Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest that phosphorylation signaling precedes and regulates mechanosensing during focal adhesion formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.29.518437v1?rss=1 Authors: Kasanga, E., Han, Y., Shifflet, M., Navarrete, W., McManus, R., Parry, C., Barhona, A., Nejtek, V., Richardson, J. R., Salvatore, M. F. Abstract: Mechanisms that augment dopamine (DA) signaling to compensate for tyrosine hydroxylase (TH) loss and delay motor impairment in Parkinsons disease remain unidentified. The rat nigrostriatal pathway was unilaterally-lesioned by 6-OHDA to determine whether differences in DA content, TH protein, TH phosphorylation, or D1 receptor expression in striatum or substantia nigra (SN) aligned with onset of hypokinesia at two time points. At 7 days, DA and TH loss in striatum exceeded 95%, whereas DA was unaffected in SN, despite 60% TH loss. At 28 days, hypokinesia was established. At both time points, ser31 TH phosphorylation increased only in SN, corresponding to less DA versus TH loss. ser40 TH phosphorylation was unaffected in striatum or SN. By day 28, D1 receptor expression increased only in lesioned SN. These results indicate that increased ser31 TH phosphorylation and D1 receptor in the SN, not striatum, augment DA signaling against TH loss to mitigate hypokinesia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.14.516427v1?rss=1 Authors: Li, T. Y., Wang, Q., Gao, A. W., Li, X., Mottis, A., Shong, M., Auwerx, J. Abstract: Lysosomes are central platforms for not only the degradation of macromolecules but also the integration of multiple signaling pathways. However, whether and how lysosomes mediate the mitochondrial stress response (MSR) remain largely unknown. Here, we demonstrate that lysosomal acidification via the vacuolar H+-ATPase (v-ATPase) is essential for the transcriptional activation of the mitochondrial unfolded protein response (UPRmt). Mitochondrial stress stimulates v-ATPase-mediated lysosomal activation of the mechanistic target of rapamycin complex 1 (mTORC1), which then directly phosphorylates the MSR transcription factor, activating transcription factor 4 (ATF4). Disruption of mTORC1-dependent ATF4 phosphorylation blocks the UPRmt, but not other similar stress responses, such as the UPRER. Finally, ATF4 phosphorylation downstream of the v-ATPase/mTORC1 signaling is indispensable for sustaining mitochondrial redox homeostasis and protecting cells from reactive oxygen species (ROS)-associated cell death upon mitochondrial stress. Thus, v-ATPase/mTORC1-mediated ATF4 phosphorylation via lysosomes links mitochondrial stress to UPRmt activation and mitochondrial function resilience. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In this week's episode we review a novel approach to generating autologous CD7-specific CAR T therapy for patients with T-cell malignancies that overcomes a key limitation: target-driven fratricide. We'll also learn about new research pinpointing a key subset of exhausted CD4+ T cells in B- ALL, which also provides a rationale for combining tyrosine kinase inhibitors and PD-L1 blockers to reverse exhaustion and enhance leukemia clearance. Finally, we'll discuss studies in a mouse model of acute ischemic stroke, showing that inhibiting phosphorylation of a tight junction protein in endothelial cells reduces risk of intracranial hemorrhage after treatment with recombinant tissue plasminogen activator.

References Dr. Guerra's notes The Journal of Nutrition, Volume 137, Issue 3, March 2007, Pages 548–553 --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message

References Dr Guerra's synthesis of the relevant literature BioEssays, Volume: 39, Issue: 5, First published: 20 February 2017. Front Cardiovasc Med. 2020; 7: 2. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message

References Dr Guerra's pathbiochemistry lecture notes Cancer Res. 2011 May 1; 71(9): 3400–3409 --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message

References Dr Guerra lecture notes Front. Endocrinol., 14 October 2014. Sci Rep. 2020; 10: 21628. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support

My AP Biology Thoughts  Unit 4 Cell Communication and Cell CycleWelcome to My AP Biology Thoughts podcast, my name is Corrinna and I am your host for episode #88. This is Unit 4 Cell Communication and Cell Cycle and today, we will be talking about transduction phosphorylation cascades Segment 1: Introduction to transduction: phosphorylation cascadesTransduction is the second step in cell signaling pathways. It comes after reception, where the signal (which is called the ligand) is received by the receptor.  In order for the signal to start a response in the protein, the receptor needs to be activated. For the cell to produce a response, the next proteins in the chain also need to be activated. These proteins can be activated and deactivated like an on/off switch.  One of the ways that the signaling molecules are activated is phosphorylation. For a molecule to be phosphorylated, phosphate is added to the molecule. Phosphate groups are typically linked to either tyrosine, threonine, or serine, since these amino acids have hydroxyl groups in their side chains.  Phosphorylation is what can activate or deactivate the signaling molecules. It can also make the proteins more active (like an enzyme) or cause it to be broken down. Additionally, phosphorylation generally isn't permanent. To de-phosphorylate a protein, cells have enzymes called phosphatases that remove the phosphate groups from the phosphorylated protein.  A phosphorylation cascade is when multiple signaling molecules in the cell signaling chain are phosphorylated, which transports the signal to another molecule to produce the end result.  Segment 2: examples of transduction: phosphorylation cascadesIn order to better understand phosphorylation cascades, let's look at an example.  One example of a phosphorylation cascade is the epidermal growth factor (EGF) pathway.  When growth factor ligands bind to the receptors, the receptors act as kinases and attach phosphate groups to each other's intracellular tails. These receptors are now activated, triggering a series of events. Since these events don't include phosphorylation, we won't cover them in detail and will instead talk about the parts after that series that do involve phosphorylation.  Those events activate kinase Raf. This activated Raf phosphorylates and activates MEK, which in turn phosphorylates and activates ERKs. The ERKs then phosphorylate and activate other target molecules that then promote cell growth and division.  This specific pathway is called a mitogen-activated protein kinase cascade.  Because this specific pathway used multiple phosphorylation events that triggered other phosphorylations, it can be classified as a phosphorylation cascade.  Segment 3: Connection to the Course Phosphorylation cascades are extremely important in cell signaling pathways because they allow the cell to respond to more than one cell signal. Phosphorylation cascades trigger multiple cellular responses, because the phosphorylation of one protein leads to the phosphorylation of another.  Additionally, if phosphorylation cascades become out of control, especially cascades that signal for growth factor, cancer can occur. This shows that being able to stop cell signaling is extremely important, since if cell growth and division goes unregulated, it becomes dangerous.  To stop cell growth and division, the cell may receive a signal to undergo apoptosis, or cell death. This usually happens if a cell doesn't pass a checkpoint in the cell cycle, which is explained in further detail in another episode.  Thank you for listening to this episode of My AP Biology Thoughts. For more student-run podcasts and digital content, make sure that you visit http://www.hvspn.com (www.hvspn.com). See you next time on My AP Biology thoughts Podcast! Music Credits: "Ice Flow" Kevin MacLeod (incompetech.com) Licensed under...

My AP Biology Thoughts  Unit 4 Cell Communication and Cell CycleWelcome to My AP Biology Thoughts podcast, my name is Victoria Villagran and I am your host for episode #90 called Unit 4 Cell Communication and Cell Cycle: Cellular Responses in Signaling Pathways. Today we will be discussing general cellular responses in signaling pathways.  Segment 1: Introduction to Cellular Responses in Signaling Pathways Signaling pathway: Signaling begins with the recognition of a chemical messenger, a ligand, by a receptor protein in a target cell The ligand-binding domain of a receptor recognizes a specific chemical messenger, which can be a peptide, a small chemical, or protein, in a specific one-to-one relationship Signal reception, which is when the target cell receives a signaling molecule; transduction, which is a series of events that converts the signal to something the target cell can respond to; and cellular response, which is when the target cell responds to the signal.  They are many varieties of ligands and receptors, and them binding can lead to many different responses  May involved 2nd messengers or phosphorylation, and protein phosphorylation  They all produce some kind of cellular response The same signal or ligand can lead to different responses  Signal Pathways may influence how the cell responds to its environment  Responses can be short or long term We can see changes such as an increase in the transcription of certain genes or the activity of particular enzymes. We may be able to see changes in the outward behavior or appearance of the cell, such as cell growth or cell death, that are caused by the molecular changes Three Response Types 1. Metabolism/Growth/Enzyme Activation/Open Ion Channel Altering the activity of specific enzymes, metabolic enzymes in the cell become more or less active  Activated G Protein binds to a molecule that starts the transduction pathway, we will go into more detail later  Some signals bind to ligand-gated ion channels which either open or close in response to binding  2. Gene Expression The process where information from a gene is used by the cell to produce a functional product, a protein. It involves two steps, transcription and translation. Signaling pathways can target either or both steps to alter the amount of a particular protein produced in a cell. Intracellular receptor: ligands are small or non-polar and can diffuse into the membrane. They are an activated hormone-receptor that can act as a transcription factor and affect gene expression Controlling which genes are expressed or not through a number control mechanisms  3. Apoptosis: programmed cell death in cell cycle (also T cell Recognition) Internal signals (such as those triggered by damaged DNA) can lead to apoptosis, but so can signals from outside the cell.  Segment 2: More About Specific Cellular Responses  Metabolism: Epinephrine  When epinephrine binds to its receptor on a muscle cell (G protein-coupled receptor), it triggers a signal transduction cascade involving production of the second messenger molecule cyclic AMP (cAMP). This cascade leads to phosphorylation of two metabolic enzymes, causing a change in the enzymes' behavior. The first enzyme is glycogen phosphorylase (GP) which breaks down glycogen into glucose. Phosphorylation activates glycogen phosphorylase, causing lots of glucose to be released. The second enzyme that gets phosphorylated is glycogen synthase (GS) and it is involved in building up glycogen, and phosphorylation inhibits its activity.  Through regulation of these enzymes, a muscle cell rapidly gets a large, ready pool of glucose molecules. The glucose is available for use by the muscle cell in response to a sudden surge of adrenaline aka the “fight or flight” response. Apoptosis: External Signaling  Most animal cells have receptors that interact with the...

My AP Biology Thoughts  Unit 3 Cellular EnergeticsWelcome to My AP Biology Thoughts podcast, my name is Jacqueline Sun and I am your host for episode #5 called Unit 3 Cellular Energetics: OXIDATIVE PHOSPHORYLATION. Today we will be discussing the formation of ATP in the vital final step of cellular respiration.  Segment 1: Introduction to Oxidative Phosphorylation Oxidative Phosphorylation is the name for the entire final process in which ATP is created; it can be split into two parts which are the ETC and chemiosmosis.  Is an aerobic process which requires oxygen and follows the Krebs cycle. Uses 3 NADH and 1 FADH2 produced from the Krebs cycle, also requires oxygen as the final electron acceptor. The final product of Oxidative Phosphorylation is, of course, ATP. A byproduct is H2O. Takes place in the inner membrane of the mitochondria, involves 4 protein complexes labeled with roman numerals 1-4 from left to right and an ATP synthase that are all embedded within the membrane. Segment 2: More About Oxidative PhosphorylationETC There are two electron carriers which will start the ETC: NADH and FADH2. I will first talk about NADH, because the process for FADH2 is slightly different. The electron carrier NADH is oxidized into NAD+, losing two electrons which are pumped into protein I. This energy transfer allows one H+ ion (lost from the NADH) to be actively transported into the intermembrane space. The H+ must be actively transported because there is a higher concentration of H+ in the intermembrane space than in the matrix. The electrons are then transferred by a transfer protein to protein III , and the energy that is lost in the transfer is again used to pump an H+ ion into the intermembrane space. Finally the lower-energy level electrons are transported once more to protein IV, and the energy lost in the transfer is used to pump another H+ over. At this point, the 2 electrons are much lower in energy level and must be removed to prevent a backup of electrons, so they will exit the last protein complex and bind with two free-flowing H+ ions and ½ of an O2 molecule to create H2O. This is how oxygen acts as the final electron acceptor and how it contributes to the creation of the byproduct of H2O. In summary, by the end of the process, NADH has pumped 3 total electrons. Now let's talk about FADH2. FADH2 is the other electron carrier and is oxidized at protein II, losing two electrons to be pumped into the ETC and turning into FAD and two H+ ions. It will then follow the same process as NADH. The key difference here is that FADH2 starts at protein II while NADH starts at protein I, meaning FADH2 will only pump two protons, making it slightly less efficient than NADH.  Chemiosmosis (compared to previous process, considerably more straightforward) Overall, ATP synthase, the central protein complex of chemiosmosis, will convert the potential energy of the proton gradient into chemical energy in ATP. Due to the ETC, there is a higher concentration of protons in the intermembrane space and lower conc. in the matrix, the protons in the higher conc. will naturally want to move down their electrochemical gradient into the matrix. The H+ ions will pass through the ATP synthase back into the matrix. The energy derived from the movement of these protons down their gradient and through the ATP synthase allows for phosphorylation, or the binding of Phosphate to ADP, to occur. From this, we have finally created ATP, and the cycle of cellular respiration is complete. Segment 3: Connection to the Course Oxidative Phosphorylation is extremely important, for it is the last component of cellular respiration, one of the most important life functions that generates an organism's energy in the form of ATP. Without this energy, I would not be speaking here right now, and all life in general would cease. Large quantities of ATP cannot be created without this process, but at the same time Oxidative

My AP Biology Thoughts   Unit 3 Cellular EnergeticsWelcome to My AP Biology Thoughts podcast, my name is Chloe McGregor and I am your host for episode #65 called Unit 3 Cellular Energetics: Enzyme activators and inhibitors. Today we will be discussing the role of activators and inhibitors in enzyme activity.  Segment 1: Introduction to activators and inhibitors  It is important to understand the enzyme substrate complex first. Substrates and Enzymes are extremely specific to one another regarding the shape and charge of the site at which they bond. This site is called the active site. Each different substrate utilizes a specific enzyme, and is broken down from there. However, enzyme activity can be altered by both inhibitors and activators. More specifically, inhibitors are often disadvantageous as they decrease the rate at which the enzyme can work. On the other hand, activators work in order to turn enzymes back on so they can work. There are so many processes going on in the body which are not all happening at the same time. Because of this, enzymes are often deactivated, and not at work. This is why activators are so important because they activate the enzyme in order for it to function again.  Segment 2: More About Enzyme activators and inhibitors  To go more in depth, there are 2 types of enzyme inhibitors. Competitive inhibitors attach to the active site of the enzyme which prevents the substrate from binding to the enzyme. This is known as competitive inhibition because the substrate is competing with the inhibitor for the active site. To combat this, you can increase the substrate concentration in order to ultimately outcompete the inhibitors. Another type of inhibitor is the noncompetitive inhibitor. This is when it attaches to a different site on the enzyme causing the enzyme to change shape which prevents any substrate from binding at all. Another word for this is allosteric regulation. This inhibition can not be combatted because the enzyme changes shape, so increasing the substrate concentration would have no impact at all. In addition to these common inhibitors, feedback inhibition is when the final product of a reaction acts as an inhibitor to the first enzyme. This shuts down the pathway, and can be done either competitively or noncompetitively. There are also two branches of activators. Allosteric activation is when a new molecule binds to a different site on the enzyme to open the active site back up. Phosphorylation, on the other hand, happens when the existing protein structure is chemically modified. An example of this can be shifting a hydrophobic region to a hydrophilic region to allow certain reactions to occur. Phosphorylation also opens back up the active site similar to allosteric activation, however there is no outside molecule.  Segment 3: Connection to the Course Inhibitors and activators are important in the greater scheme of things. In terms of cell energetics, both cellular respiration and photosynthesis are metabolic pathways. This means that there are a ton of tiny steps involved each utilizing different enzymes. In terms of activators, it is important that enzymes are activated and ready to catalyze a reaction when it is time. Oxidative phosphorylation is a great example of enzyme activation which is so crucial in order to make a sufficient amount of ATP. In terms of inhibitors, they are important for regulating metabolic pathways. Inhibitors can be beneficial as they stop the overproduction of products when there is no need for them. This is evident in the glycolytic pathway through allosteric regulation. Protein structure is also also crucial to the way a cell does work, so inhibiting an enzyme can cause a domino effect as the tertiary and quaternary structures begin to unfold. Overall, activators and inhibitors are important factors when studying enzyme activity, and it is important to understand their effects on the catalysis...