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1. Compound Mutations in the Abl1 Kinase Cause Inhibitor Resistance by Shifting DFG Flip Mechanisms and Relative State Populations

   https://doi.org/10.1101/2024.05.23.595569  

   Monteiro_da_Silva, Gabriel; Lam, Kyle; Dalgarno, David C; Rubenstein, Brenda M (May 2024, bioRxiv)

   Abstract The intrinsic dynamics of most proteins are central to their function. Protein tyrosine kinases such as Abl1 undergo significant conformational changes that modulate their activity in response to different stimuli. These conformational changes constitute a conserved mechanism for self-regulation that dramatically impacts kinases’ affinities for inhibitors. Few studies have attempted to extensively sample the pathways and elucidate the mechanisms that underlie kinase inactivation. In large part, this is a consequence of the steep energy barriers associated with many kinase conformational changes, which present a significant obstacle for computational studies using traditional simulation methods. Seeking to bridge this knowledge gap, we present a thorough analysis of the “DFG flip” inactivation pathway in Abl1 kinase. By leveraging the power of the Weighted Ensemble methodology, which accelerates sampling without the use of biasing forces, we have comprehensively simulated DFG flip events in Abl1 and its inhibitor-resistant variants, revealing a rugged landscape punctuated by potentially druggable intermediate states. Through our strategy, we successfully simulated dozens of uncorrelated DFG flip events distributed along two principal pathways, identified the molecular mechanisms that govern them, and measured their relative probabilities. Further, we show that the compound Glu255Lys/Val Thr315Ile Abl1 variants owe their inhibitor resistance phenotype to an increase in the free energy barrier associated with completing the DFG flip. This barrier stabilizes Abl1 variants in conformations that can lead to loss of binding for Type-II inhibitors such as Imatinib or Ponatinib. Finally, we contrast our Abl1 observations with the relative state distributions and propensity for undergoing a DFG flip of evolutionarily-related protein tyrosine kinases with diverging Type-II inhibitor binding affinities. Altogether, we expect that our work will be of significant importance for protein tyrosine kinase inhibitor discovery, while also furthering our understanding of how enzymes self-regulate through highly-conserved molecular switches. 

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2. Pseudophosphatases as Regulators of MAPK Signaling

   https://doi.org/10.3390/ijms222212595  

   Hepworth, Emma Marie; Hinton, Shantá D. (November 2021, International Journal of Molecular Sciences)

   Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes, including the cell cycle, apoptosis, differentiation, protein biosynthesis, and oncogenesis; therefore, tight control of the activity of MAPK is critical. Kinases and phosphatases are well established as MAPK activators and inhibitors, respectively. Kinases phosphorylate MAPKs, initiating and controlling the amplitude of the activation. In contrast, MAPK phosphatases (MKPs) dephosphorylate MAPKs, downregulating and controlling the duration of the signal. In addition, within the past decade, pseudoenzymes of these two families, pseudokinases and pseudophosphatases, have emerged as bona fide signaling regulators. This review discusses the role of pseudophosphatases in MAPK signaling, highlighting the function of phosphoserine/threonine/tyrosine-interacting protein (STYX) and TAK1-binding protein (TAB 1) in regulating MAPKs. Finally, a new paradigm is considered for this well-studied cellular pathway, and signal transduction pathways in general. 

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3. Inhibition of AKT Signaling Alters βIV Spectrin Distribution at the AIS and Increases Neuronal Excitability

   https://doi.org/10.3389/fnmol.2021.643860  

   Di Re, Jessica; Hsu, Wei-Chun J.; Kayasandik, Cihan B.; Fularczyk, Nickolas; James, T. F.; Nenov, Miroslav N.; Negi, Pooran; Marosi, Mate; Scala, Federico; Prasad, Saurabh; et al (June 2021, Frontiers in Molecular Neuroscience)

   null (Ed.)

   The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing βIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on βIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in βIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders. 

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4. Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment

   https://doi.org/10.3389/fmed.2023.1146115  

   Mijit, Mahmut; Boner, Megan; Cordova, Ricardo A; Gampala, Silpa; Kpenu, Eyram; Klunk, Angela J; Zhang, Chi; Kelley, MarK R; Staschke, Kirk A; Fishel, Melissa L (April 2023, Frontiers in Medicine)

   Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment. 

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5. Peroxisomal Stress Response and Inter-Organelle Communication in Cellular Homeostasis and Aging

   https://doi.org/10.3390/antiox11020192  

   Kim, J; Bai, H (January 2022, Antioxidants)

   Santos, AL (Ed.)

   Peroxisomes are key regulators of cellular and metabolic homeostasis. These organelles play important roles in redox metabolism, the oxidation of very-long-chain fatty acids (VLCFAs), and the biosynthesis of ether phospholipids. Given the essential role of peroxisomes in cellular homeostasis, peroxisomal dysfunction has been linked to various pathological conditions, tissue functional decline, and aging. In the past few decades, a variety of cellular signaling and metabolic changes have been reported to be associated with defective peroxisomes, suggesting that many cellular processes and functions depend on peroxisomes. Peroxisomes communicate with other subcellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum (ER), and lysosomes. These inter-organelle communications are highly linked to the key mechanisms by which cells surveil defective peroxisomes and mount adaptive responses to protect them from damages. In this review, we highlight the major cellular changes that accompany peroxisomal dysfunction and peroxisomal inter-organelle communication through membrane contact sites, metabolic signaling, and retrograde signaling. We also discuss the age-related decline of peroxisomal protein import and its role in animal aging and age-related diseases. Unlike other organelle stress response pathways, such as the unfolded protein response (UPR) in the ER and mitochondria, the cellular signaling pathways that mediate stress responses to malfunctioning peroxisomes have not been systematically studied and investigated. Here, we coin these signaling pathways as “peroxisomal stress response pathways”. Understanding peroxisomal stress response pathways and how peroxisomes communicate with other organelles are important and emerging areas of peroxisome research. 

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