Endogenous Inhibitor Protein Research Articles

Paradoxical action of PP2A inhibition and its potential for therapeutic sensitization.

The protein phosphatase 2A (PP2A), a serine/threonine phosphatase, is recognized as a tumor suppressor involved in diverse cellular processes and essential for maintaining cell viability in vivo. However, endogenous inhibitors of PP2A such as cancerous inhibitor of PP2A (CIP2A) and endogenous nuclear protein inhibitor 2 of PP2A (SET) counteract the anticancer function of PP2A, promoting tumorigenesis, development, and drug resistance in tumors. Surprisingly though, contrary to conventional understanding, inhibition of the tumor suppressor gene PP2A with exogenous small molecule compounds can enhance the efficacy of cancer treatment and achieve superior tumor inhibition. Moreover, exogenous PP2A inhibitors resensitize cancers to treatment and provide novel therapeutic strategies for drug-resistant tumors, which warrant further investigation.

NLRP3/1-mediated pyroptosis: beneficial clues for the development of novel therapies for Alzheimer's disease.

The inflammasome is a multiprotein complex involved in innate immunity that mediates the inflammatory response leading to pyroptosis, which is a lytic, inflammatory form of cell death. There is accumulating evidence that nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome-mediated microglial pyroptosis and NLRP1 inflammasome-mediated neuronal pyroptosis in the brain are closely associated with the pathogenesis of Alzheimer's disease. In this review, we summarize the possible pathogenic mechanisms of Alzheimer's disease, focusing on neuroinflammation. We also describe the structures of NLRP3 and NLRP1 and the role their activation plays in Alzheimer's disease. Finally, we examine the neuroprotective activity of small-molecule inhibitors, endogenous inhibitor proteins, microRNAs, and natural bioactive molecules that target NLRP3 and NLRP1, based on the rationale that inhibiting NLRP3 and NLRP1 inflammasome-mediated pyroptosis can be an effective therapeutic strategy for Alzheimer's disease.

Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

PHI-1, an Endogenous Inhibitor Protein for Protein Phosphatase-1 and a Pan-Cancer Marker, Regulates Raf-1 Proteostasis.

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1's inhibitory potency. Inhibiting PP1 to mimic PHI-1's function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.

MiR-125a-5p/miR-125b-5p contributes to pathological activation of angiotensin II-AT1R in mouse distal convoluted tubule cells by the suppression of Atrap

The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.

Abstract 1100: miR205 mediates acquired resistance to ALK inhibition via targeting Mig6 expression and enhancing EGFR signaling

Abstract Complete responses to ALK tyrosine kinase inhibitors (TKIs) are rare and resistance eventually develops in ALK fusion-positive non-small cell lung cancer patients. To overcome resistance and improve therapeutic outcomes, it is crucial to understand the molecular mechanisms contributing to resistance. Our lab has previously shown EGFR signaling mediates adaptive resistance to ALK inhibitors. We demonstrated that RNA and protein levels of Mig6, an endogenous protein inhibitor of EGFR, were suppressed rapidly following ALK inhibition, thus unlocking EGFR from inhibition to support cell survival. In this study, we asked whether EGFR signaling activation and Mig6 suppression persist once acquired resistance is established. An EML4-ALK cell line, H3122, was continuously exposed to a fixed dose of the ALK inhibitor crizotinib to generate three resistant lines (H3122-CR1, -2, and -3). All H3122-CR lines lacked ALK kinase mutations and were also cross-resistant to other ALK TKIs including ceritinib and alectinib. We found phosphorylation and total EGFR were upregulated while Mig6 protein was attenuated across all resistant lines compared to their parental counterpart. Afatinib, a pan-ERBB family inhibitor, or Mig6 overexpression, was able to re-sensitize those resistant cells to ALK inhibition. Interestingly, we did not find Mig6 overexpression altered phosphorylation of EGFR. Previously reported data suggest that Mig6 competes with Shc1, a critical signaling adapter shared by ALK and EGFR, for the same substrate-binding cleft on EGFR. We then hypothesized Mig6 could block EGFR-Shc1 binding and downstream signaling transduction without directly impacting EGFR phosphorylation. Indeed, the co-immunoprecipitation assay showed Mig6 knockdown in H3122 cells enhanced Shc1 binding to EGFR without altering phosphorylation of EGFR itself, suggesting a novel mechanism in regulating EGFR signaling by impairing the signaling adapter binding. We then investigated the mechanism responsible for Mig6 protein attenuation in resistant lines. By examining the RNA-seq data for all the CR lines compared to the parental H3122, we found MIR205HG was substantially upregulated in resistant cells. MIR205HG is the host gene for miR-205, which was known to target ERRFI1 gene that encodes Mig6. Stem-loop RT-qPCR confirmed that miR-205 was increased ~5 fold in all CR lines. Overexpressing miR-205 in H3122 cells could downregulate Mig6 expression in a dose-dependent manner. Herein we presented a novel resistance mechanism to ALK inhibition by which miR205 upregulation attenuates Mig6 expression, releasing EGFR-Shc1 signaling transduction from inhibition to support cell survival. This study also provides additional support for targeting EGFR signaling to overcome ALK TKI resistance to improve patient survival. Citation Format: Nan Chen, Robert C. Doebele. miR205 mediates acquired resistance to ALK inhibition via targeting Mig6 expression and enhancing EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1100.

Stop CRYing! Inhibition of cryptochrome function by small proteins.

Plants can detect the presence of light using specialised photoreceptor proteins. These photoreceptors measure the intensity of light, but they can also respond to different spectra of light and thus ‘see' different colours. Cryptochromes, which are also present in animals, are flavin-based photoreceptors that enable plants to detect blue and ultraviolet-A (UV-A) light. In Arabidopsis, there are two cryptochromes, CRYPTOCHROME 1 (CRY1) and CRYPTOCHROME 2 (CRY2) with known sensory roles. They function in various processes such as blue-light mediated inhibition of hypocotyl elongation, photoperiodic promotion of floral initiation, cotyledon expansion, anthocyanin production, and magnetoreception, to name a few. In the dark, the cryptochromes are in an inactive monomeric state and undergo photochemical and conformational change in response to illumination. This results in flavin reduction, oligomerisation, and the formation of the ‘cryptochrome complexome'. Mechanisms of cryptochrome activation and signalling have been extensively studied and found to be conserved across phylogenetic lines. In this review, we will therefore focus on a far lesser-known mechanism of regulation that is unique to plant cryptochromes. This involves inhibition of cryptochrome activity by small proteins that prevent its dimerisation in response to light. The resulting inhibition of function cause profound alterations in economically important traits such as plant growth, flowering, and fruit production. This review will describe the known mechanisms of cryptochrome activation and signalling in the context of their modulation by these endogenous and artificial small inhibitor proteins. Promising new applications for biotechnological and agricultural applications will be discussed.

Exploring the factors that affect the themostability of barley limit dextrinase – Inhibitor complex

Barley Limit dextrinase (Hordeum vulgare HvLD) is the unique endogenous starch-debranching enzyme, determining the production of a high degree of fermentation. The activity of HvLD is regulated by an endogenous LD inhibitor protein (LDI). In beer production, free LD is easy to inactivate in mashing process under the condition of high temperature. The binding of LD with LDI protects it against heat inactivation. Exploring the factors affecting the themostability of HvLD-LDI complex is important for beer production. In this work, the themostability of HvLD-LDI complex at different NaCl concentrations and temperatures were explored by molecular dynamics simulation and binding free energy calculation. In NaCl solution, the complex exhibits higher conformational stability at 343 K and 363 K than those in pure water. Root mean square fluctuation (RMSF) analysis identified the thermal sensitive regions of HvLD and LDI. The binding free energy results suggest that the LD-LDI complex is more stable in NaCl solution than those in pure water at high temperature. The residues with high contribution to the complex were identified. The structural and dynamic details will help us to understand the driving forces that lead to the themostability of HvLD-LDI complex at different temperatures and different salt concentrations, which will facilitate the optimization conditions of beer production for maintaining the thermal stability and activity of HvLD.

Interaction mechanism of endogenous PP2A inhibitor protein ENSA with PP2A.

The vast diversity of protein phosphatase 2A (PP2A) holoenzyme composition ensures its multifaceted role in the regulation of cellular growth and signal transduction. In several pathological conditions, such as cancer, PP2A is inhibited by endogenous inhibitor proteins. Several PP2A inhibitor proteins have been identified, one of which is α-endosulfine (ENSA). ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. The role of ENSA in PP2A inhibition is rather well characterized, but knowledge of the mechanism of inhibition is scarce. In this study, we have performed comprehensive structural characterization of ENSA, and its interaction with PP2A A- and various B56-subunit isoforms by combining NMR spectroscopy, small-angle X-ray scattering (SAXS) and interaction assays. The results clearly indicate that ENSA is an intrinsically disordered protein containing three transient α-helical structures. ENSA was observed to interact PP2A mainly via A-subunit, as the affinity with the A-subunit is significantly stronger than with any of the B56 subunits. Based on our results, it seems that ENSA follows the dock-and-coalesce mechanism in associating with PP2A A-subunit. Taken together, our results provide an essential structural and molecular framework to understanding molecular bases of ENSA-mediated PP2A inhibition, which is crucial for the development of new therapies for diseases linked to PP2A inhibition.

The crystal structure of a 250-kDa heterotetrameric particle explains inhibition of sheddase meprin β by endogenous fetuin-B

Meprin β (Mβ) is a multidomain type-I membrane metallopeptidase that sheds membrane-anchored substrates, releasing their soluble forms. Fetuin-B (FB) is its only known endogenous protein inhibitor. Herein, we analyzed the interaction between the ectodomain of Mβ (MβΔC) and FB, which stabilizes the enzyme and inhibits it with subnanomolar affinity. The MβΔC:FB crystal structure reveals a ∼250-kDa, ∼160-Å polyglycosylated heterotetrameric particle with a remarkable glycan structure. Two FB moieties insert like wedges through a "CPDCP trunk" and two hairpins into the respective peptidase catalytic domains, blocking the catalytic zinc ions through an "aspartate switch" mechanism. Uniquely, the active site clefts are obstructed from subsites S4 to S10', but S1 and S1' are spared, which prevents cleavage. Modeling of full-length Mβ reveals an EGF-like domain between MβΔC and the transmembrane segment that likely serves as a hinge to transit between membrane-distal and membrane-proximal conformations for inhibition and catalysis, respectively.

The Interaction Mechanism of Intrinsically Disordered PP2A Inhibitor Proteins ARPP-16 and ARPP-19 With PP2A.

Protein phosphatase 2A (PP2A) activity is critical for maintaining normal physiological cellular functions. PP2A is inhibited by endogenous inhibitor proteins in several pathological conditions including cancer. A PP2A inhibitor protein, ARPP-19, has recently been connected to several human cancer types. Accordingly, the knowledge about ARPP-19—PP2A inhibition mechanism is crucial for the understanding the disease development and the therapeutic targeting of ARPP-19—PP2A. Here, we show the first structural characterization of ARPP-19, and its splice variant ARPP-16 using NMR spectroscopy, and SAXS. The results reveal that both ARPP proteins are intrinsically disordered but contain transient secondary structure elements. The interaction mechanism of ARPP-16/19 with PP2A was investigated using microscale thermophoresis and NMR spectroscopy. Our results suggest that ARPP—PP2A A-subunit interaction is mediated by linear motif and has modest affinity whereas, the interaction of ARPPs with B56-subunit is weak and transient. Like many IDPs, ARPPs are promiscuous binders that transiently interact with PP2A A- and B56 subunits using multiple interaction motifs. In summary, our results provide a good starting point for future studies and development of therapeutics that block ARPP-PP2A interactions.

Sphingosine Analogs and Protein Phosphatase 2A as a Molecular Targeted Cancer Therapy: A Mini Systematic Review

Background: Regulation of protein phosphatase 2A (PP2A) plays an important role in hematologic and solid neoplasms. Therefore, the use of sphingosine analogs as anti-neoplastic drugs has shown potential due to their role as PP2A activators. Objective: Investigation of whether sphingosine analogs bind to endogenous inhibitor proteins of PP2A, such as I2 PP2A/SET and/or CIP2A, and whether this binding reactivates PP2A, allowing it to resume its role as a tumor suppressor. Methods: Literature from the PubMed database was searched and those articles related to PP2A and sphingosine analogs were reviewed. Results: Utilization of sphingosine analogs in hematologic and solid neoplasms revealed numerous mechanisms of inducing cell death. Regulation of PP2A through modulation of I2 PP2A/SET and/or CIP2A was demonstrated in a variety of neoplastic processes; however, unique mechanisms such as cell necrosis via the production of reactive oxygen species was also appreciated. Conclusion: Only certain malignancies expressed endogenous inhibitor proteins, yet sphingosine analogs were able to induce cell death in neoplasms that did not express these proteins. This suggests that sphingosine analogs may be utilized for anti-neoplastic therapy via reactivation of PP2A however, it is not the exclusive mechanism for inducing cell death. Further investigation of sphingosine analogs as a novel or adjunctive chemotherapeutic treatment is warranted.

Arpp19 Promotes Myc and Cip2a Expression and Associates with Patient Relapse in Acute Myeloid Leukemia.

Disease relapse from standard chemotherapy in acute myeloid leukemia (AML) is poorly understood. The importance of protein phosphatase 2A (PP2A) as an AML tumor suppressor is emerging. Therefore, here, we examined the potential role of endogenous PP2A inhibitor proteins as biomarkers predicting AML relapse in a standard patient population by using three independent patient materials: cohort1 (n = 80), cohort2 (n = 48) and The Cancer Genome Atlas Acute Myeloid Leukemia (TCGA LAML) dataset (n = 160). Out of the examined PP2A inhibitors (CIP2A, SET, PME1, ARPP19 and TIPRL), expression of ARPP19 mRNA was found to be independent of the current AML risk classification. Functionally, ARPP19 promoted AML cell viability and expression of oncoproteins MYC, CDK1, and CIP2A. Clinically, ARPP19 mRNA expression was significantly lower at diagnosis (p = 0.035) in patients whose disease did not relapse after standard chemotherapy. ARPP19 was an independent predictor for relapse both in univariable (p = 0.007) and in multivariable analyses (p = 0.0001) and gave additive information to EVI1 expression and risk group status (additive effect, p = 0.005). Low ARPP19 expression was also associated with better patient outcome in the TCGA LAML cohort (p = 0.019). In addition, in matched patient samples from diagnosis, remission and relapse phases, ARPP19 expression was associated with disease activity (p = 0.034), indicating its potential usefulness as a minimal residual disease (MRD) marker. Together, these data demonstrate the oncogenic function of ARPP19 in AML and its risk group independent role in predicting AML patient relapse tendency.

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1.

The mitochondrial adenosine triphosphate (ATP) synthase produces most of the ATP required by mammalian cells. We isolated porcine tetrameric ATP synthase and solved its structure at 6.2-angstrom resolution using a single-particle cryo-electron microscopy method. Two classical V-shaped ATP synthase dimers lie antiparallel to each other to form an H-shaped ATP synthase tetramer, as viewed from the matrix. ATP synthase inhibitory factor subunit 1 (IF1) is a well-known in vivo inhibitor of mammalian ATP synthase at low pH. Two IF1 dimers link two ATP synthase dimers, which is consistent with the ATP synthase tetramer adopting an inhibited state. Within the tetramer, we refined structures of intact ATP synthase in two different rotational conformations at 3.34- and 3.45-Å resolution.

ATP production under lockdown

Structural Biology Cellular processes must respond to change, often by speeding up, slowing down, or stopping altogether. Adenosine triphosphate (ATP) synthases use a transmembrane proton gradient to produce ATP, but this reaction can go in reverse and needs to be halted when conditions are unfavorable. Jinke Gu et al. purified a tetrameric ATP synthase complex from pig hearts that contained the endogenous inhibitory protein IF1. Targeted refinement yielded high-resolution views of the mammalian ATP synthase trapped in two different rotation states by IF1. The findings suggest that ATP synthase tetramers can be inhibited by at least three different mechanisms. Science , this issue p. [1068][1] [1]: /lookup/doi/10.1126/science.aaw4852

Adaptation and diversification of venomous snake proteins

According to the World Health Organization, approximately 1.8–2.7 million people worldwide suffer from venomous snake bites each year and at least 138,000 of these incidents are fatal. Whereas, snakebite envenoming poses a serious threat to public health, yet the snake venom toxins endow several pharmacological effects, including presynaptic neurotoxicity, myotoxicity, and cardiotoxicity, as well as anticoagulant, hemolytic, hemorrhagic, edema-inducing, and platelet aggregation-inhibiting effects. Duplication and mutation of the genes encoding these toxins play an important role in generating molecular diversity. Curiously, venomous snakes are not lethal to the viper itself because the viper’s resistance against its own venom. However, endogenous inhibitor proteins evolutionarily acquired by venomous snakes to protect themselves have not yet been fully characterised because it is unclear how to inhibit for target toxin due to the lack of information including mutation analysis and the three-dimensional structures of the inhibitors. This review provides an overview of endogenous inhibitors of venomous snake as regulation systems for the toxin proteins. Recently, we isolated some inhibitors targeting different toxins from the sera of the Japanese vipers. We investigated the evolution of these endogenous inhibitors, which have been significantly influenced by positive selection. Directional mutagenesis, where mutation hotspots are found in genes encoding molecular surface proteins and functional domains of these proteins, acts as a diversifying mechanism for the exquisite biological targeting necessary to protect the host from its own venom.

Cellular and pathophysiological consequences of Arp2/3 complex inhibition: role of inhibitory proteins and pharmacological compounds.

The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.

Inhibition of the chimeric DnaJ‐PKAc enzyme by endogenous inhibitor proteins

The chimeric DnaJ-PKAc enzymeresulting from an approximately 400-kb deletion of chromosome 19 is a primary contributor to the oncogenic transformation that occurs in fibrolamellar hepatocellular carcinoma, also called fibrolamellar carcinoma (FLC). This oncogenic deletion juxtaposes exon 1 of the DNAJB1 heat shock protein gene with exon 2 of the PRKACA gene encoding the protein kinase A catalytic subunit, resulting in DnaJ-PKAc fusion under the transcriptional control of the DNAJB1 promoter. The expression of DnaJ-PKAc is approximately 10 times that of wild-type (wt) PKAc catalytic subunits, causing elevated and dysregulated kinase activity that contributes to oncogenic transformation. In normal cells, PKAc activity is regulated by a group of endogenous proteins, termed protein kinase inhibitors (PKI) that competitively inhibit PKAc and assist with the nuclear export of the enzyme. Currently, it is scarcely known whether interactions with PKI are perturbed in DnaJ-PKAc. In this report, we survey existing data sets to assess the expression levels of the various PKI isoforms that exist in humans to identify those that are candidates to encounter DnaJ-PKAc in both normal liver and FLC tumors. We then compare inhibition profiles of wtPKAc and DnaJ-PKAc against PKI and demonstrate that extensive structural homology in the active site clefts of the two enzymes confers similar kinase activities and inhibition by full-length PKI and PKI-derived peptides.

SET SUMOylation promotes its cytoplasmic retention and induces tau pathology and cognitive impairments

PP2A is a major regulator of tau phosphorylation, which is principally regulated by an endogenous nuclear protein inhibitor 2 of PP2A (I2PP2A), also named SET. However, how SET is post-translationally regulated and translocates from the nucleus to the cytoplasm remain incompletely understood. Here we show SET is SUMOylated at K68 residue that induces its cytoplasmic retention, resulting in Alzheimer disease (AD) like tau pathology and cognitive defects. SET is predominantly SUMOylated at K68 that leads to its translocation from the nucleus to the cytoplasm and subsequently induces inhibition of PP2A and hyperphosphorylation of tau in HEK-293 cells. Moreover, overexpression of wild type SET significantly inhibits PP2A activity, leading to tau hyperphosphorylation, less synapse loss and cognitive deficits. Conversely, blocking SET SUMOylation via mutating Lys 68 to Arg rescues tau pathology and cognitive impairments in C57/BL6 mice infected with adeno-associated virus encoding SET. Further, β-amyloid exposure of rat primary hippocampal neurons induces a dose-dependent SUMOylation of SET. Our findings suggest that SET SUMOylation stimulates its cytoplasmic retention and inhibits PP2A activity, consequently leading to tau hyperphosphorylation and cognitive impairments, which provides a new insight into the AD-like tau pathology.