ZDHHC5: a pivotal palmitoyltransferase orchestrating signaling networks - unraveling mechanisms and therapeutic horizons.

Membrane Association and Targeting of Prenylated Ras-like GTPases

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Simple SummaryCircular RNAs (circRNAs) are single-stranded RNA molecules that form a covalently closed loop structure. They are characterized by distinct features and multifariously implicated in the regulation of both physiological and pathological states. The aberrant expression of circRNAs has been evidenced in various malignancies. Circular transcripts can effectively modulate gene expression. The most prevalent manner through which circRNAs promote cancer development and progression is their interaction with key components of major signaling pathways. In particular, abnormally expressed circRNAs can dictate the crosstalk between signaling cascades. In recent years, there has been great progress regarding circRNA research in the context of cancer progression, and various regulatory axes have been described. As our knowledge of signaling regulation by circRNAs continuously expands, novel therapeutic approaches can be assessed and established, seeking to overcome clinical challenges, such as the treatment of cancer patients with distant metastasis and those who relapse.Signal transduction is an essential process that regulates and coordinates fundamental cellular processes, such as development, immunity, energy metabolism, and apoptosis. Through signaling, cells are capable of perceiving their environment and adjusting to changes, and most signaling cascades ultimately lead to alterations in gene expression. Circular RNAs (circRNAs) constitute an emerging type of endogenous transcripts with regulatory roles and unique properties. They are stable and expressed in a tissue-, cell-, and developmental stage-specific manner, while they are involved in the pathogenesis of several diseases, including cancer. Aberrantly expressed circRNAs can mediate cancer progression through regulation of the activity of major signaling cascades, such as the VEGF, WNT/β-catenin, MAPK, PI3K/AKT, and Notch signaling pathways, as well as by interfering with signaling crosstalk. Deregulated signaling can then function to induce angiogenesis, promote invasion, migration, and metastasis, and, generally, modulate the hallmarks of cancer. In this review article, we summarize the most recently described and intriguing cases of circRNA-mediated signaling regulation that are involved in cancer progression, and discuss the biomarker potential of circRNAs, as well as future therapeutic applications.

The development of artificial intelligence (AI) technology has made significant contributions to the healthcare field, especially in the development of innovative drugs and therapies. The combination of computational sophistication and AI data analysis has enabled researchers to identify complex patterns in biomedical data, accelerate drug discovery time, and facilitate therapy personalization. This research aims to explore the important role of AI in drug development and innovative therapies to create a better future of healthcare. This involves an analysis of various AI methods and techniques used in drug development as well as the application of AI in personalized therapy for society. This study was conducted by conducting a literature review and analyzing the latest research and developments in the application of AI in drug and therapy development. The results showed that AI has opened new opportunities in drug development by accelerating the process of drug target identification, molecular simulation, and optimization of clinical trials. Meanwhile, in therapeutics, AI enables better personalization through analysis of patient clinical data and prediction of response to specific treatments. This opens up the potential for the development of more effective and targeted therapies. With the development of AI technology, the development of innovative drugs and therapies has become more efficient and effective. The application of AI in healthcare offers the potential to create a more personalized, precise, and comprehensive healthcare future. The collaboration between medical science and AI technology will lead to more innovative and affordable health solutions for the people. Thus, the role of AI in the development of innovative drugs and therapies is recognized as one of the key pillars in creating a better future of healthcare.

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.

Crossing Membranes

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.

Kinases are firmly established drug targets in cancer. There are currently 44 FDA approved kinase drug and hundreds of compounds are in clinical development. However, less than 10% of the Kinome is currently targeted and a large proportion is considered understudied by the NIH Illuminating the Druggable Genome Program (https://druggablegenome.net/). No small molecule inhibitors are known for these "dark" proteins, yet many may be opportune novel cancer targets.We developed a computational pipeline to identify and prioritize understudied kinases as cancer drug targets. We analyzed the complete set of tumors in The Cancer Genome Atlas (TCGA). For 33 different cancers we performed differential expression analysis and identified 39 dark kinases that exhibit significant upregulation in at least four types. Using co-expression analysis we built functional networks prioritizing drug targets. To identify small molecules that reverse their expression levels, we leveraged transcriptional response signatures obtained from dozens of human cancer cell lines exposed to tens of thousands of small molecules from the Library of Integrated Network-based Cellular Signatures (LINCS). To identify small molecules that directly bind to and inhibit dark kinases, we have have combined an advanced AI (artificial intelligence) model trained on activity data from across the Kinome with structure-based simulations.Using the computational pipeline, we identified the dark Ca2+/Calmodulin dependent kinase PNCK as the most differentially overexpressed kinase in kidney cancer patients. Our analyses have demonstrated statistically significant correlation between PNCK mRNA levels and various clinical and pathological outcomes, including histologic grade, clinical staging and overall survival. We have confirmed high levels of PNCK expression in 5 renal cell carcinoma cell lines (Caki-1, ACHN, 786-O, A704 and A498). Knockdown and overexpression studies have suggested PNCK and the CaMK pathway may contribute to cellular proliferation and cell cycle progression. We have applied our AI-based screening pipeline to a library of >20 million commercially available compounds and confirmed three PNCK inhibiting chemotypes. In summary, using a novel computational pipeline, we have identified and experimentally validated PNCK as a prospective novel drug target in an understudied pathway that is highly upregulated in kidney cancer. We identified first in class small molecules that target this previously dark kinase as prospective starting points for optimization into a clinical candidate.Citation Format: Derek J. Essegian, Rimpi Khurana, Vasileios Stathias, Valery Chavez, Jaime R. Merchan, Stephan Schürer. The dark cancer kinome - untapped opportunities for the development of novel drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5103.

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.

Endothelial nitric-oxide synthase (eNOS) function is fundamentally modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (bovine)/1177 (human) by Akt has been shown to be the central mechanism of eNOS regulation. Here we revealed a novel role of proteasome in controlling eNOS serine 1179 phosphorylation and function. Rather than affecting eNOS turnover, proteasomal inhibition specifically dephosphorylated eNOS serine 1179, leading to decreased enzymatic activity. Blocking protein phosphatase 2A (PP2A) by okadaic acid or PP2A knockdown restored eNOS serine 1179 phosphorylation and activity in proteasome-inhibited cells. Although total PP2A expression and activity in cells were not affected by proteasome inhibitors, proteasomal inhibition induced PP2A ubiquitination and ubiquitinated PP2A translocated from cytosol to membrane. Further biochemical analyses demonstrated that eNOS associated with PP2A on cell membranes. Proteasomal inhibition markedly enhanced PP2A association to eNOS, and this increase of PP2A dephosphorylated eNOS and its upstream kinase Akt. Taken together, these studies identified a novel pathway in which proteasome modulates eNOS phosphorylation by inducing intracellular PP2A translocation.

Molecular disturbances of neurotransmitter systems have long been held to be a core feature of the pathophysiology of schizophrenia. Despite years of study of neurotransmitter associated protein expression at multiple levels of gene expression, reports of abnormal neurotransmitter receptor transcript, protein, and signaling complex expression in schizophrenia brain have often been conflicting. These inconsistencies led us to reconsider neurotransmitter-based hypotheses of schizophrenia not as a problem of receptor number, or as a defect of neurotransmitter systems, but rather as a dysregulation of central cellular processes regulating the intracellular distribution of signaling proteins. Our working hypothesis is that a fundamental dysregulation of intracellular processes exists in schizophrenia, resulting in abnormal assembly, trafficking, and intracellular targeting of many key proteins involved in neurotransmission and other critical cellular functions. Previous studies have shown the important roles posttranslational lipid and carbohydrate modifications play in targeting receptors, transporters, and other proteins between intracellular compartments and the synapse, and in the lateral translocation of such molecules between lipid microdomains at the distal end of forward trafficking pathways. Accordingly, we have predicted that abnormal posttranslational lipid and/or sugar modification of proteins by occurs in schizophrenia. We have previously reported changes in extent of N-linked glycosylation as well as of the lipid modifications palmitoylation and N-myristoylation on target proteins in schizophrenia brain. In an ongoing project to elucidate mechanisms of these changes, we have studied expression patterns of key enzymes associated with these posttranslational modifications. Using well characterized samples of postmortem brain from schizophrenia and matched comparison subjects, we assayed transcript expression of enzymes associated with posttranslational protein modifications by lipids and carbohydrates using microarrays and qPCR. Next, we assayed protein expression of a subset of enzymes using western blot analyses. To determine the brain cell specificity of protein changes, we used laser capture microdissection (LCM) of neuronal and glial cells to harvest specific cell populations from postmortem brains, and developed and validated a capillary electrophoresis system for ultra-low quantity protein concentration (the ProteinSimple WES system) to measure protein expression within LCM harvested cells. Using microarray and qPCR, multiple transcript changes were found in schizophrenia cortex. The most substantial number of altered transcripts were found for those encoding enzymes associated with multiple aspects of posttranslational carbohydrate modifications of proteins, including N-acetylglucosaminyltransferases (GlcNAcTs), glucosyltransferases, glucosidases, N-acetylgalactosaminyltransferases (GalNAcTs), galactosyltransferases, mannosidases, fucosyltransferases, fucosidases, sialyltransferases, and sialidases. Fewer changes were found for lipid modification enzymes. Subsequently, protein expression of candidate proteins associated with these posttranslational modifications were determined by western blot analyses. Changes in protein expression of mutlple enzymes associated with glycan modification of proteins were found in schizophrenia. These include significant decreases in expression of the N-acetylglucosaminyltransferases B3GNT8 and MGAT4A, and the fucosyltransferase FUT8. Increased protein expression was found for the fucosyltransferase POFUT2, the sialyltransferase ST8SIA2, the glucosyltransferase UGGT2, and the mannosidase EDEM2. Numerous protein changes were also found in enzymes associated with lipid and glycolipid modifications. These include decreased expression of the prenylation associate denzyme subunits farnesyl-protein transferase α-subunit (FNTA), geranylgeranyltransferase type I β-subunit (PGGT1B), and rab geranylgeranyltransferase β-subunit (RABGGTB). Glycophosphatidylinositol (GPI)-anchor attachment 1 protein (GPAA1) is increased in these subjects. To determine the cell-specific pattern of these protein changes, we have developed an LCM-capillary electrophoresis assay to isolate protein from LCM harvested cells to allow multiplex protein quantification in 500 ng of protein obtained from these cells. We have validated that we can reliably harvest cortical neuronal subtypes and astroglia, are able to measure 4 proteins simultaneously in samples from these cells lines, and are currently collecting cells to extend these findings into cell-specific studies to determine if the changes we have found in posttranslational modification proteins are widely specific or specific to given subpopulations of brain cells. These data support our earlier findings of altered patterns of the posttranslational modifications of both glycosylation and lipid modification of proteins in the cortex of schizophrenia. By identifying changes in both mRNA and protein expression of key enzymes associated with these posttranslational modifications, we have begun to elucidate potential mechanisms of these earlier observations. One of the challenges that has plagued schizophrenia research for decades is that many different neurotransmitter and neurochemical systems have been implicated and studied in this illness, and reconciling this large literature is challenging. These many changes in numerous different systems suggest, however, that rather than schizophrenia being a disorder of a given neurotransmitter system, it is rather a disturbance of core intracellular processes that underlie regulation of multiple neurochemical systems. The machinery associated with posttranslational modifications of proteins is a possible substrate that could reconcile prior abnormalities identified in myriad systems. We have proposed that a fundamental defect in the brains of those affected with this illness is abnormal assembly, trafficking and receptor dynamics of many different proteins in schizophrenia that is due mechanistically to abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of lipid and glycan modification of proteins are likely candidates for such a process, and these present data begin to elucidate the mechanisms from which these abnormalities occur.

<p>Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is a major obstacle in the success of head and neck cancer therapy. Despite efforts by several groups to understand the mechanisms of resistance to tyrosine kinase inhibitors such as erlotinib, there has been little success in improving the patient survival. Given that there are a number of ongoing clinical trials testing the efficacy of erlotinib in head and neck cancer, it is essential to investigate the novel mechanisms of erlotinib resistance to improve its efficacy and patient survival. This dissertation addresses this issue of erlotinib resistance in head and neck cancer, underscoring the role of inflammatory cytokine signaling. <strong>Chapter 1</strong> introduces the problem of erlotinib resistance and discusses the potential link between inflammatory signaling and cancer progression and erlotinib resistance in head and neck squamous cell carcinoma. <strong>Chapter 2</strong> discusses the role of the cytokine interleukin-6 signaling in acquired resistance to erlotinib in head and neck squamous cell carcinoma. <strong>Chapter 3</strong> describes the role of IL-1 signaling in acquired resistance to erlotinib in head and neck squamous cell carcinoma. <strong>Chapter 4</strong> discusses the specific role of IL-1α (an agonistic ligand for IL-1 signaling) in acquired resistance to erlotinib in head and neck squamous cell carcinoma. <strong>Chapter 5</strong> discusses ideas to test for future work in this field. Altogether, this dissertation endeavors to emphasize the contributory role of inflammatory cytokine signaling in erlotinib resistance in head and neck squamous cell carcinoma so that it helps in the development of effective anti-cancer therapies and biomarkers of resistance and/or response in HNSCC.</p>

The field of inositol signaling has expanded greatly in recent years. Given the many reviews on phosphoinositide kinases, we have chosen to restrict our discussion to inositol lipid hydrolysis focused on the phosphatases and a brief mention of the lipase isoforms. We also discuss recent discoveries that link mutations in phosphoinositide phosphatases to disease.

c-Jun N-terminal kinase (JNK) signalling regulates both cancer cell apoptosis and survival. Emerging evidence show that JNK promoted tumour progression is involved in various cancers, that include human pancreatic-, lung-, and breast cancer. The pro-survival JNK oncoprotein functions in a cell context- and cell type-specific manner to affect signal pathways that modulate tumour initiation, proliferation, and migration. JNK is therefore considered a potential oncogenic target for cancer therapy. Currently, designing effective and specific JNK inhibitors is an active area in the cancer treatment. Some ATP-competitive inhibitors of JNK, such as SP600125 and AS601245, are widely used in vitro; however, this type of inhibitor lacks specificity as they indiscriminately inhibit phosphorylation of all JNK substrates. Moreover, JNK has at least three isoforms with different functions in cancer development and identifying specific selective inhibitors is crucial for the development of targeted therapy in cancer. Some selective inhibitors of JNK are identified; however, their clinical studies in cancer are relatively less conducted. In this review, we first summarised the function of JNK signalling in cancer progression; there is a focus on the discussion of the novel selective JNK inhibitors as potential targeting therapy in cancer. Finally, we have offered a future perspective of the selective JNK inhibitors in the context of cancer therapies. We hope this review will help to further understand the role of JNK in cancer progression and provide insight into the design of novel selective JNK inhibitors in cancer treatment.

Maintaining a functional proteome under different environmental conditions is challenging for every organism, in particular for unicellular organisms, such as bacteria. In order to cope with changing environments and stress conditions, bacteria depend on strictly coordinated proteostasis networks that control protein production, folding, trafficking, and degradation. Regulation of ribosome biogenesis and protein synthesis are cornerstones of this cellular adaptation in all domains of life, which is rationalized by the high energy demand of both processes and the increased resistance of translationally silent cells against internal or external poisons. Reduced protein synthesis ultimately also reduces the substrate load for protein transport systems, which are required for maintaining the periplasmic, inner, and outer membrane subproteomes. Consequences of impaired protein transport have been analyzed in several studies and generally induce a multifaceted response that includes the upregulation of chaperones and proteases and the simultaneous downregulation of protein synthesis. In contrast, generally less is known on how bacteria adjust the protein targeting and transport machineries to reduced protein synthesis, e.g., when cells encounter stress conditions or face nutrient deprivation. In the current review, which is mainly focused on studies using Escherichia coli as a model organism, we summarize basic concepts on how ribosome biogenesis and activity are regulated under stress conditions. In addition, we highlight some recent developments on how stress conditions directly impair protein targeting to the bacterial membrane. Finally, we describe mechanisms that allow bacteria to maintain the transport of stress-responsive proteins under conditions when the canonical protein targeting pathways are impaired.