Regulation Of Protein Activity Research Articles

Expression of Cystathionine-β-Synthase (CBS) Genes in Grape under Various Abiotic Stresses and Subcellular Localization of VvCBS27

The cystathionine-β-synthase (CBS) domain is present in the proteins of all living organisms and functions as an energy sensor, regulating protein activity through the binding capacity of its adenosine ligands. The role of the CBS gene in plant growth and development, as well as tolerance to abiotic stresses, remains largely unknown, especially in grapevine. In our study, 32 members of the CBS gene family were obtained that were distributed on 15 chromosomes. The results of the structural and evolutionary tree analyses indicated that the VvCBS gene family exhibits diverse intron-exon patterns and highly conserved motifs. Furthermore, the phylogenetic classification of the VvCBS genes revealed the presence of three subfamilies. Subcellular localization analyses showed that the VvCBS genes are mainly located in the plasma membrane region. The secondary structure of the VvCBS protein mainly consists of α-helices, extended strands, β-turns, and random coils. The VvCBS gene family exhibited four co-linear gene pairs, while the numbers for Arabidopsis thaliana and rice were 21 and 7, respectively. The promoter cis-acting element analysis revealed the presence of light-responsive, hormone-responsive, stress-responsive, and growth- and development-related elements in the VvCBS family. The expression characterization demonstrated that 12 VvCBS genes exhibited high expression levels in all grape tissues. Additionally, the RT-qPCR expression analyses showed that the 32 VvCBS exhibited different responses to a variety of abiotic stresses (cold, drought, salt), suggesting that they were functionally differentiated. VvCBS27 was cloned from ‘Pinot Noir’ of grapevine with a coding sequence of 624 bp. Subcellular localization showed that VvCBS27 protein was mainly located in the cytoplasm, cell membrane, and nucleus. This study lays a foundation for elucidating the function of grape CBS protein.

Posttranslational modifications: an emerging functional layer of diet-host-microbe interactions.

The microbiome plays a vital role in human health, with changes in its composition impacting various aspects of the body. Posttranslational modification (PTM) regulates protein activity by attaching chemical groups to amino acids in an enzymatic or non-enzymatic manner. PTMs offer fast and dynamic regulation of protein expression and can be influenced by specific dietary components that induce PTM events in gut microbiomes and their hosts. PTMs on microbiome proteins have been found to contribute to host-microbe interactions. For example, in Escherichia coli, S-sulfhydration of tryptophanase regulates uremic toxin production and chronic kidney disease in mice. On a broader microbial scale, the microbiomes of patients with inflammatory bowel disease exhibit distinct PTM patterns in their metaproteomes. Moreover, pathogens and commensals can alter host PTM profiles through protein secretion and diet-regulated metabolic shifts. The emerging field of metaPTMomics focuses on understanding PTM profiles in the microbiota, their association with lifestyle factors like diet, and their functional effects on host-microbe interactions.

Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90β, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1−/− cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

Sealing the Pandora’s vase of pancreatic fistula through entrapping the digestive enzymes within a dextrorotary (D)-peptide hydrogel

A variety of therapeutic possibilities have emerged for skillfully regulating protein function or conformation through intermolecular interaction modulation to rectify abnormal biochemical reactions in diseases. Herein, a devised strategy of enzyme coordinators has been employed to alleviate postoperative pancreatic fistula (POPF), which is characterized by the leakage of digestive enzymes including trypsin, chymotrypsin, and lipase. The development of a dextrorotary (D)-peptide supramolecular gel (CP-CNDS) under this notion showcases its propensity for forming gels driven by intermolecular interaction. Upon POPF, CP-CNDS not only captures enzymes from solution into hydrogel, but also effectively entraps them within the internal gel, preventing their exchange with counterparts in the external milieu. As a result, CP-CNDS completely suppresses the activity of digestive enzymes, effectively alleviating POPF. Remarkably, rats with POPF treated with CP-CNDS not only survived but also made a recovery within a mere 3-day period, while mock-treated POPF rats had a survival rate of less than 5 days when experiencing postoperative pancreatic fistula, leak or abscess. Collectively, the reported CP-CNDS provides promising avenues for preventing and treating POPF, while exemplifying precision medicine-guided regulation of protein activity that effectively targets specific pathogenic molecules across multiple diseases.

Thermal stability of bivalent cation/phosphoinositide domains in model membranes

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.

Post-Translational Modification of PTEN Protein: Quantity and Activity.

Post-translational modifications play crucial roles in regulating protein functions and stabilities. PTEN is a critical tumor suppressor involved in regulating cellular proliferation, survival, and migration processes. However, dysregulation of PTEN is common in various human cancers. PTEN stability and activation/suppression have been extensively studied in the context of tumorigenesis through inhibition of the PI3K/AKT signaling pathway. PTEN undergoes various post-translational modifications, primarily including phosphorylation, acetylation, ubiquitination, SUMOylation, neddylation, and oxidation, which finely tune its activity and stability. Generally, phosphorylation modulates PTEN activity through its lipid phosphatase function, leading to altered power of the signaling pathways. Acetylation influences PTEN protein stability and degradation rate. SUMOylation has been implicated in PTEN localization and interactions with other proteins, affecting its overall function. Neddylation, as a novel modification of PTEN, is a key regulatory mechanism in the loss of tumor suppressor function of PTEN. Although current therapeutic approaches focus primarily on inhibiting PI3 kinase, understanding the post-translational modifications of PTEN could help provide new therapeutic strategies that can restore PTEN's role in PIP3-dependent tumors. The present review summarizes the major recent developments in the regulation of PTEN protein level and activity. We expect that these insights will contribute to better understanding of this critical tumor suppressor and its potential implications for cancer therapy in the future.

SUMO1 modification of 0N4R-tau is regulated by PIASx, SENP1, SENP2, and TRIM11

Tau is a microtubule-associated protein that contributes to cytoskeletal stabilization. Aggregation of tau proteins is associated with neurodegenerative disorders such as Alzheimer's disease. Several types of posttranslational modifications that alter the physical properties of tau proteins have been identified. SUMOylation is a reversible modification of lysine residues by a small ubiquitin-like modifier (SUMO). In this study, we examined the enzymes that regulate the SUMOylation and deSUMOylation of tau in an alternatively spliced form, 0N4R-tau. Among SUMO E3 ligases, we found protein inhibitor of activated STAT (PIAS)xα and PIASxβ increase the levels of SUMOylated tau. The deSUMOylation enzymes sentrin-specific protease (SENP)1 and SENP2 reduced the levels of SUMO-conjugated tau. SUMO1 modification increased the level of phosphorylated tau, which was suppressed in the presence of SENP1. Furthermore, we examined the effect of tripartite motif (TRIM)11, which was recently identified as an E3 ligase for SUMO2 modification of tau. We found that TRIM11 increased the modification of both 2N4R- and 0N4R-tau by SUMO1, which was attenuated by mutation of the target lysine residue to arginine. These findings suggest that the expression and activity of SUMOylation regulatory proteins modulate the physical properties of tau proteins and may contribute to the onset and/or progression of tau-associated neurodegenerative disorders.

CoDIAC: A comprehensive approach for interaction analysis reveals novel insights into SH2 domain function and regulation.

Protein domains are conserved structural and functional units and are the functional building blocks of proteins. Evolutionary expansion means that domain families are often represented by many members in a species, which are found in various configurations with other domains, which have evolved new specificity for interacting partners. Here, we develop a structure-based interface analysis to comprehensively map domain interfaces from available experimental and predicted structures, including interfaces with other macromolecules and intraprotein interfaces (such as might exist between domains in a protein). We hypothesized that a comprehensive approach to contact mapping of domains could yield new insights. Specifically, we use it to gain information about how domains selectivity interact with ligands, whether domain-domain interfaces of repeated domain partnerships are conserved across diverse proteins, and identify regions of conserved post-translational modifications, using relationship to interaction interfaces as a method to hypothesize the effect of post-translational modifications (and mutations). We applied this approach to the human SH2 domain family, an extensive modular unit that is the foundation of phosphotyrosine-mediated signaling, where we identified a novel approach to understanding the binding selectivity of SH2 domains and evidence that there is coordinated and conserved regulation of multiple SH2 domain binding interfaces by tyrosine and serine/threonine phosphorylation and acetylation, suggesting that multiple signaling systems can regulate protein activity and SH2 domain interactions in a regulated manner. We provide the extensive features of the human SH2 domain family and this modular approach, as an open source Python package for COmprehensive Domain Interface Analysis of Contacts (CoDIAC).

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

Inhibitory effect of Curcumin on a cervical cancer cell line via the RAS/RAF signaling pathway.

Cervical cancer has a very important place in female infertility and ranks fourth among cancers affecting women. Curcumin (CUR) is closely associated with the expression and activity of various regulatory proteins. It is also known that curcumin has preventive and therapeutic effects on various types of cancer. In this study, the anticancer activities of curcumin were demonstrated in the human cervical cancer cell line (HeLa). qRT-PCR and western blot analyses were used to evaluate mRNA and protein expression of curcumin in HeLa and immortalized human skin keratinocyte cell lines (HaCaT) (proliferation and apoptosis regulatory markers of the RAS/RAF signaling pathway). MTT analysis was performed, showing HeLa and HaCaT cell proliferation depending on the dose and duration of curcumin and doxorubicin. A wound scratch healing assay was applied to examine cell migration and invasion of HeLa after curcumin application. To determine the role of curcumin and doxorubicin in the apoptosis of HeLa cells, the mRNA levels of caspase-3 were examined by qRT-PCR. The results were analyzed with a one-way ANOVA SPSS 20.0 program. CUR (IC50: 242.8 μM) and DOX (IC50: 92.1 μM) were determined to have the ability to inhibit the proliferation of HeLa cells and induce apoptosis over a 72-hour period and dose-dependently. Moreover, the results revealed that the mRNA and protein expression levels of RAF and RAS in HeLa cells were downregulated by CUR and DOX. The findings show that an alternative treatment method for cervical cancer can be developed with the application of CUR and DOX. Alternative methods for cervical cancer treatment may be developed using different methods in future studies.

Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer.

To identify drivers of malignancy in human pancreatic ductal adenocarcinoma (PDAC), we performed regulatory network analysis on a large collection of expression profiles from laser capture microdissected samples of PDAC and benign precursors. We discovered that BMAL2 plays a role in the initiation, progression, post resection survival, and KRAS activity in PDAC. Functional analysis of BMAL2 target genes led us to hypothesize that it plays a role in regulating the response to hypoxia, a critical but poorly understood feature of PDAC physiology. Knockout of BMAL2 in multiple human PDAC cell lines revealed effects on viability and invasion, particularly under hypoxic conditions. Loss of BMAL2 also affected glycolysis and other metabolic processes. We found that BMAL2 directly regulates hypoxia-responsive target genes. We also found that BMAL2 is necessary for the stabilization of HIF1A upon exposure to hypoxia, but destabilizes HIF2A under hypoxia. These data demonstrate that BMAL2 is a master transcriptional regulator of hypoxia responses in PDAC and may serve as a long-sought molecular switch that distinguishes HIF1A- and HIF2A-dependent modes of hypoxic metabolism.

Aptamer-Based DNA Allosteric Switch for Regulation of Protein Activity.

Allostery is a fundamental way to regulate the function of biomolecules playing crucial roles in cell metabolism and proliferation and is deemed the second secret of life. Given the limited understanding of the structure of natural allosteric molecules, the development of artificial allosteric molecules brings a huge opportunity to transform the allosteric mechanism into practical applications. In this study, the concept of bionics is introduced into the design of artificial allosteric molecules and an allosteric DNA switch with an activity site and an allosteric site based on two aptamers for selective inhibition of thrombin activity. Compared with the single aptamer, the allosteric switch possesses a significantly enhanced inhibition ability, which can be precisely regulated by converting the switch states. Moreover, the dynamic allosteric switch is further subjected to the control of the DNA threshold circuit for realizing automatic concentration determination and activity inhibition of thrombin. These compelling results confirm that this allosteric switch equipped with self-sensing and information-processing modules puts a new slant on the research of allosteric mechanisms and further application of allosteric tactics in chemical and biomedical fields.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W–S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Evaluating the Dynamics of Cyp3a11, MRP2 and MDR1 Modulation by IL-22

Background: IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing. In addition, IL-22 reduces the expression of MRP2 and MDR1, the gene that encodes as Multidrug resistance protein 2 or 1 involve in the drug detoxification. Modulation of these proteins can influence the oral bioavailability of drugs. Aims: The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11, MRP2 and MDR1 expression and protein level or activity in the intestinal epithelium. Methods: In vitro enteroids of the mouse small intestine were cultured in 3D in the presence/absence of IL-22 for 5 days. Cyp3a11, MRP2 and MDR1 transcript expression was evaluated by qPCR followed by protein analysis to define their modulation and activity at varying timepoints after IL-22 treatment cessation. Results: IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. This action of IL-22 are made without any modulation of protein level of Cyp3a11. In the same way, IL-22 reduces the expression and the protein level of MRP2 and MDR1, an effect normalizes after treatment cessation. Conclusions: IL-22 suppresses the expression and the activity of Cyp3a11 without any modulation of the protein level unlike at the level of MRP2 and MDR1 which are reduced. These data suggest that regulation of gene expression, protein or enzyme activity by IL-22 may have variable effects: Cyp3a11 is ATP-independent, whereas MRP2 and MDR1 are ATP-dependent. This direct or indirect enzymatic modulation of these proteins must be taken into account when assessing the role of the drug in the biology of the intestinal mucosa. Dr. Lloyd Sutherland Chair in IBD/GI Research/Weston Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

C-di-AMP determines the hierarchical organization of bacterial RCK proteins

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

SUMOylation of zebrafish transcription factor Zbtb21 affects its transcription activity.

Post-translational modification by Small Ubiquitin-like MOdifier (SUMO) is an important mechanism to regulate protein activity, protein stability, and localization of substrates. Zbtb21 is a zinc finger and BTB (Broad-complex, Tram-track and Bric à brac) domain-containing transcription factor. Bioinformatic prediction suggests several putative SUMOylated sites in Zbtb21 protein. Two evolutionarily conserved lysine residues in Zbtb21 protein were mutated alone or in combination to disrupt the binding with SUMO molecules. Western blot and co-immunoprecipitation analyses were performed to detect the SUMOylation state of wild type and mutant Zbtb21 proteins, respectively. Luciferase reporter assays were conducted to evaluate their transcription activities. Meanwhile, immunofluorescence staining was carried out to show their sub-nuclear localizations. Finally, co-immunoprecipitation was performed to detect the interaction between Zbtb21 and its partners. Phylogenetically conserved lysines 419 and 845 of zebrafish Zbtb21 protein can be conjugated with SUMO molecules. SUMOylation does not affect the subcellular localization and protein stability of Zbtb21, as well as the interaction with Zbtb14 or Zbtb21. Nevertheless, luciferase reporter assays revealed that Zbtb21 is a dual-function transcription factor which exerts activation or repression effect on different promoters, and SUMOylation can modulate the transcriptional activity of Zbtb21 in regulating downstream target genes. Hence, Zbtb21 is identified as a novel substrate of SUMOylation, which would be important for its function. Zebrafish Zbtb21 protein can be SUMOylated on lysines 419 and 845, which is evolutionary conserved. SUMOylation affects the dual role of Zbtb21 on transcription.

Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer.

The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6516.