Specific Serine Research Articles

Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B

BackgroundEpigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.MethodsKDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.ResultsRSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.ConclusionsOverall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.

Aberrant cytoplasmic expression of UHRF1 restrains the MHC-I-mediated anti-tumor immune response

Immunotherapy successfully complements traditional cancer treatment. However, primary and acquired resistance might limit efficacy. Reduced antigen presentation by MHC-I has been identified as potential resistance factor. Here we show that the epigenetic regulator ubiquitin-like with PHD and ring finger domains 1 (UHRF1), exhibits altered expression and aberrant cytosolic localization in cancerous tissues, where it promotes MHC-I ubiquitination and degradation. Cytoplasmic translocation of UHRF1 is induced by its phosphorylation on a specific serine in response to signals provided by factors present in the tumor microenvironment (TME), such as TGF-β, enabling UHRF1 to bind MHC-I. Downregulation of MHC-I results in suppression of the antigen presentation pathway to establish an immune hostile TME. UHRF1 inactivation by genetic deletion synergizes with immune checkpoint blockade (ICB) treatment and induces an anti-tumour memory response by evoking low-affinity T cells. Our study adds to the understanding of UHRF1 in cancer immune evasion and provides a potential target to synergize with immunotherapy and overcome immunotherapeutic resistance.

RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.

Cellular sex throughout the organism underlies somatic sexual differentiation

Sex chromosomes underlie the development of male or female sex organs across species. While systemic signals derived from sex organs prominently contribute to sex-linked differences, it is unclear whether the intrinsic presence of sex chromosomes in somatic tissues has a specific function. Here, we use genetic tools to show that cellular sex is crucial for sexual differentiation throughout the body in Drosophila melanogaster. We reveal that every somatic cell converts the intrinsic presence of sex chromosomes into the active production of a sex determinant, a female specific serine- and arginine-rich (SR) splicing factor. This discovery dismisses the mosaic model which posits that only a subset of cells has the potential to sexually differentiate. Using cell-specific sex reversals, we show that this prevalence of cellular sex drives sex differences in organ size and body weight and is essential for fecundity. These findings demonstrate that cellular sex drives differentiation programs at an organismal scale and highlight the importance of cellular sex pathways in sex trait evolution.

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete, but abnormally activated in a wide variety of tumors. The CTA, Testis specific serine kinase 6 (TSSK6), is essential for male fertility in mice. Functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage independent growth, invasion and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

SUMOylation of OsPSTOL1 is essential for regulating phosphate starvation responses in rice and Arabidopsis.

Although rice is one of the main sources of calories for most of the world, nearly 60% of rice is grown in soils that are low in phosphorus especially in Asia and Africa. Given the limitations of bioavailable inorganic phosphate (Pi) in soils, it is important to develop crops tolerant to low phosphate in order to boost food security. Due to the immobile nature of Pi, plants have developed complex molecular signalling pathways that allow them to discern changes in Pi concentrations in the environment and adapt their growth and development. Recently, in rice, it was shown that a specific serine-threonine kinase known as Phosphorus-starvation tolerance 1 (PSTOL1) is important for conferring low phosphate tolerance in rice. Nonetheless, knowledge about the mechanism underpinning PSTOL1 activity in conferring low Pi tolerance is very limited in rice. Post-translation modifications (PTMs) play an important role in plants in providing a conduit to detect changes in the environment and influence molecular signalling pathways to adapt growth and development. In recent years, the PTM SUMOylation has been shown to be critical for plant growth and development. It is known that plants experience hyperSUMOylation of target proteins during phosphate starvation. Here, we demonstrate that PSTOL1 is SUMOylated in planta, and this affects its phosphorylation activity. Furthermore, we also provide new evidence for the role of SUMOylation in regulating PSTOL1 activity in plant responses to Pi starvation in rice and Arabidopsis. Our data indicated that overexpression of the non-SUMOylatable version of OsPSTOL1 negatively impacts total root length and total root surface area of rice grown under low Pi. Interestingly, our data also showed that overexpression of OsPSTOL1 in a non-cereal species, Arabidopsis, also positively impacts overall plant growth under low Pi by modulating root development. Taken together our data provide new evidence for the role of PSTOL1 SUMOylation in mediating enhanced root development for tolerating phosphate-limiting conditions.

Degradation versus fibrillogenesis, two alternative pathways modulated by seeds and glycosaminoglycans.

The mechanism that converts native human transthyretin into amyloid fibrils in vivo is still a debated and controversial issue. Commonly, non-physiological conditions of pH, temperature, or organic solvents are used in in vitro models of fibrillogenesis of globular proteins. Transthyretin amyloid formation can be achieved under physiological conditions through a mechano-enzymatic mechanism involving specific serine proteases such as trypsin or plasmin. Here, we investigate S52P and L111M transthyretin variants, both causing a severe form of systemic amyloidosis mostly targeting the heart at a relatively young age with heterogeneous phenotype among patients. Our studies on thermodynamics show that both proteins are significantly less stable than other amyloidogenic variants. However, despite a similar thermodynamic stability, L111M variant seems to have enhanced susceptibility to cleavage and a lower tendency to form fibrils than S52P in the presence of specific proteases and biomechanical forces. Heparin strongly enhances the fibrillogenic capacity of L111M transthyretin, but has no effect on the S52P variant. Fibrillar seeds similarly affect the fibrillogenesis of both proteins, with a stronger effect on the L111M variant. According to our model of mechano-enzymatic fibrillogenesis, both full-length and truncated monomers, released after the first cleavage, can enter into fibrillogenesis or degradation pathways. Our findings show that the kinetics of the two processes can be affected by several factors, such as intrinsic amyloidogenicity due to the specific mutations, environmental factors including heparin and fibrillar seeds that significantly accelerate the fibrillogenic pathway.

The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation.

Catalytic Antibodies May Contribute to Demyelination in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Here we report preliminary data demonstrating that some patients with myalgic encephalomyelitis/chronic fatiguesyndrome (ME/CFS) may have catalytic autoantibodies that cause the breakdown of myelin basic protein (MBP). We propose that these MBP-degradative antibodies are important to the pathophysiology of ME/CFS, particularly in the occurrence of white matter disease/demyelination. This is supported by magnetic resonance imagining studies that show these findings in patients with ME/CFS and could explain symptoms of nerve pain and muscle weakness. In this work, we performed a series of experiments on patient plasma samples where we isolated and characterized substrate-specific antibodies that digest MBP. We also tested glatiramer acetate (copaxone), an FDA approved immunomodulator to treat multiple sclerosis, and found that it inhibits ME/CFS antibody digestion of MBP. Furthermore, we found that aprotinin, which is a specific serine protease inhibitor, specifically prevents breakdown of MBP while the other classes of protease inhibitors had no effect. This coincides with the published literature describing catalytic antibodies as having serine protease-like activity. Postpandemic research has also provided several reports of demyelination in COVID-19. Because COVID-19 has been described as a trigger for ME/CFS, demyelination could play a bigger role in patient symptoms for those recently diagnosed with ME/CFS. Therefore, by studying proteolytic antibodies in ME/CFS, their target substrates, and inhibitors, a new mechanism of action could lead to better treatment and a possible cure for the disease.

QSAR via Multisite λ-Dynamics in the Orphaned TSSK1B Kinase.

Multisite λ-dynamics (MSλD) is a novel method for the calculation of relative free energies of binding for ligands to their targeted receptors. It can be readily used to examine a large number of molecules with multiple functional groups at multiple sites around a common core. This makes MSλD a powerful tool in structure-based drug design. In the present study, MSλD is applied to calculate the relative binding free energies of 1296 inhibitors to the testis specific serine kinase 1B (TSSK1B), a validated target for male contraception. For this system, MSλD requires significantly fewer computational resources compared to traditional free energy methods like free energy perturbation or thermodynamic integration. From MSλD simulations, we examined whether modifications of a ligand at two different sites are coupled or not. Based on our calculations, we established a quantitative structure-activity relationship (QSAR) for this set of molecules and identified a site in the ligand where further modification, such as adding more polar groups, may lead to increased binding affinity. This article is protected by copyright. All rights reserved.

Protein phosphatase 2A-B55β mediated mitochondrial p-GPX4 dephosphorylation promoted sorafenib-induced ferroptosis in hepatocellular carcinoma via regulating p53 retrograde signaling.

Rationale: As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. Methods: To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells in vitro. Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55β subunit (PP2A-B55β) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55β overexpression xenograft tumors in a nude mice model in vivo. Results: In the Sora-induced ferroptosis model of HCC in vitro, decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4Ser2) revealed that mitochondrial p-GPX4Ser2 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55β, it was found that PP2A-B55β directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55β reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55β enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Conclusion: Our data uncovered that the PP2A-B55β/p-GPX4Ser2/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4Ser2 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55β was an upstream signal modulator responsible for mitochondrial p-GPX4Ser2 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55β activation.

GALNT3 protects against vascular calcification by reducing oxidative stress and apoptosis of smooth muscle cells

Vascular calcification (VC) is the pathological deposition of calcium and phosphate minerals in blood vessels, which is a common complication of atherosclerosis. Polypeptide N-acetylgalactosamine transferase 3 (GALNT3) initiates O-glycosylation of proteins through addition of GalNAc to specific serine or threonine residues. Our previous studies revealed the potent role of GALNT3 in atherosclerosis, whereas the precise mechanisms remain obscure. This study investigated the regulatory effect and mechanism of GALNT3 on VC. Firstly, GALNT3 was overexpressed and knocked down by adenovirus in high-phosphate induced calcified HASMCs and overexpressed by adeno-associated virus in vitamin D3-induced arterial calcification mice. We showed that the calcium deposition and mRNA expression of osteogenic markers MSX2, ALPL, and Runx2 were all significantly reduced with GALNT3 overexpression. Moreover, overexpression of GALNT3 significantly down-regulated the expression of the oxidative stress markers Nox2 and Nox4, up-regulated total antioxidant capacity, decreased the expression of pro-inflammatory factors IL-1β, TNF-α and IL-8, matrix metalloproteinases MMP2 and MMP9, as well as reduced the apoptosis of cells in phosphate induced HASMCs. Furthermore, Vicia Villosa Lectin (VVL) pull down and TNFR1 immunoprecipitation assays showed that GALNT3 overexpression increased O-GalNAcylation of TNFR1 and blocked the activation of NF-κB signaling pathway. In addition, GALNT3 attenuates vitamin D3-induced aortic calcification in mice by alleviating oxidative stress and apoptosis of smooth muscle cells. In conclusion, this study indicates that GALNT3 protects against VC by reducing oxidative stress, vascular inflammation, and apoptosis of smooth muscle cells through the TNFR1/NF-κB signaling pathway. Thus, GALNT3 may be a potential therapeutic target for VC.

Phosphorylation and regulation of group II metabotropic glutamate receptors (mGlu2/3) in neurons.

Group II metabotropic glutamate (mGlu) receptors (mGlu2/3) are Gαi/o-coupled receptors and are primarily located on presynaptic axonal terminals in the central nervous system. Like ionotropic glutamate receptors, group II mGlu receptors are subject to regulation by posttranslational phosphorylation. Pharmacological evidence suggests that several serine/threonine protein kinases possess the ability to regulate mGlu2/3 receptors. Detailed mapping of phosphorylation residues has revealed that protein kinase A (PKA) phosphorylates mGlu2/3 receptors at a specific serine site on their intracellular C-terminal tails in heterologous cells or neurons, which underlies physiological modulation of mGlu2/3 signaling. Casein kinases promote mGlu2 phosphorylation at a specific site. Tyrosine protein kinases also target group II receptors to induce robust phosphorylation. A protein phosphatase was found to specifically bind to mGlu3 receptors and dephosphorylate the receptor at a PKA-sensitive site. This review summarizes recent progress in research on group II receptor phosphorylation and the phosphorylation-dependent regulation of group II receptor functions. We further explore the potential linkage of mGlu2/3 phosphorylation to various neurological and neuropsychiatric disorders, and discuss future research aimed at analyzing novel biochemical and physiological properties of mGlu2/3 phosphorylation.

Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called “NT-mini” and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT’s heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

Mass spectrometric analysis of chondroitin sulfate-linked peptides

Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3–6 monosaccharides attached to specific serine residues can be extended by 20–200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.

Abstract 1472: Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer

Abstract Nuclear factor E2-related factor 2 (NRF2) is a master regulator of cellular redox homeostasis. The sustained overactivation of NRF2 has been observed in many different types of human malignancy, conferring an advantage for growth and survival of cancer cells. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) recognizes phosphorylated serine or threonine of a target protein and isomerizes the adjacent proline residue, thereby altering the structure, stability, and function of target proteins. We identified the three specific serine residues (Ser215, 408, and 577) present in NRF2, each of which precedes proline and undergoes phosphorylation. This prompted us to investigate whether PIN1 could bind to NRF2 and thereby influence its stability. For this purpose, we initially co-transfected HEK293T cells with expression vectors for GST-PIN1 and HA-tagged wild-type Nrf2 or non-phosphorylatable mutant constructs (NRF2S215A, NRF2S408A, and NRF2S577A). Notably, there was the most pronounced ubiquitination with concomitant degradation of NRF2 in cells harbouring NRF2S215A which was associated with abrogation of its interaction with PIN1. We then investigated the clinical relevance of NRF2 and PIN1 interaction to breast cancer progression. Immunofluorescence analysis of tissue microarrays (90 breast cancer tissues and 10 adjacent normal ones) revealed much higher (P < 0.001) expression of both NRF2 and PIN1 in the invasive ductal carcinomas than in normal tissues, which correlated with disease stages. The overexpression of NRF2 and PIN1 in breast cancer patients was also confirmed by immunohistochemical and immunoblot analyses. Next, we compared the co-localization as well as expression of NRF2 and PIN1 in the two molecular subtype tissues (140 each). Both expression and co-localization of NRF2 and PIN1 were significantly higher (P < 0.001) in more aggressive TNBC than in the luminal type tissues. We also detected the significantly elevated levels of the Pin1-Nrf2 complex formed in human breast cancer specimens, compared with those in adjacent normal tissues. Silencing of Pin1 reduced the nuclear accumulation of NRF2 which was attributable to enhanced ubiquitination. The cycloheximide pulse-chase assay unravelled a significantly faster degradation of pre-existing NRF2 in PIN1-silenced MDA-MB-231 cells, corroborating the stabilization of NRF2 protein by PIN1. To explore the regulation of NRF2 by PIN1 and their interaction in vivo, we inoculated MDA-MB-231 cells into male BALB/c nude mice. Tumors derived from PIN1 knockdown cells displayed markedly reduced levels of NRF2. In conclusion, stabilization of NRF2 through direct interaction with PIN1 may provide a novel mechanism underlying their cooperative role in breast cancer growth and progression. Citation Format: Soma Saeidi, Su-Jung Kim, Yanymee N Guillen-Quispe, Achanta Sri Venkata Jagadeesh, Young-Joon Surh. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer [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 1472.

Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila.

Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). We studied the in vivo process by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). We report that many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays of the Evening activity under entraining conditions predicted the phase of activity cycles under constant darkness. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior.

Proteins of the lectin pathway of the complement system activation: immunobiological functions, genetics and involvement in the pathogenesis of human diseases

The complement system is the most ancient components in the innate immunity, mainly functioning to primarily eliminate bacterial agents intravascularly. Moreover, the complement complex proteins play a role as a bridge between the systems of innate and adaptive immunity providing adequate conditions for maturation and differentiation of B- and T-lymphocytes. The complement system consists of plasma proteins and membrane receptors. Plasma proteins interact with each other via the three described cascade pathways lectin (which is most ancient phylogenetically), alternative and classical. Lectins are proteins comprising a separate superfamily of pattern-recognizing receptors able to sense molecules of oligo- and polysaccharide nature and induce their aggregation. Among all the lectins, ficolins (FCN) (common domain fibrinogen) and collectins (common domain collagen) mannose-binding lectin (MBL), hepatic and renal collectins have exert unique functions by complexing with carbohydrate components of microbial wall. Formation of a compound complex microbial wall polysaccharides + collectin/ficolin + specific mannose-binding lectin-associated serine proteases (MARP) results in the complement system activation, inflammatory reaction and bacterium elimination. Such scenario is proceeded along the lectin pathway compared to the two other pathways called classical and alternative. Examining a role of the complement system and congenital protein defects in the pathogenesis of various diseases is of topical interest because inborn deficiency of the complement components comprises at least 5% out of total primary immunodeficiency rate, whereas the aspects of their prevalence and pathogenesis remain unexplored. Relevance of investigating the complement system components for diverse populations is tremendous, taking into consideration accumulated evidence regarding an important role of the lectin pathway in viral infections. Lectins, the main proteins in the lectin pathway of the complement activation, are encoded by polymorphic genes, wherein single nucleotide polymorphisms (SNPs) result in altered protein conformation and expression, which, in turn, affects functionality and potential to respond to a pathogen. The distribution of the lectin polymorphic gene frequencies and their haplotypes displays extremely marked population differences. According to analyzing available data, population SNP frequencies including those associated with inborn deficiencies for components of the lectin pathway have been currently scarce or unexplored. hence, here we review major lectins and their functions, their functionally significant SNPs in diverse populations and their pathogenetic importance for host defense functions.

The rate‐limiting enzyme in the CDP‐choline pathway is regulated by phosphorylation‐domain charge density

Eukaryotic cell membranes are primarily composed of phospholipids, the most abundant of which is phosphatidylcholine (PC). De novo synthesis of PC by the CDP‐choline pathway is under the control of the rate‐limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). The nuclear CCTa isoform is regulated by translocation to the inner nuclear membrane (INM) and nuclear lipid droplets (nLD) in response to lipid activators, such as oleate, or deficiency in PC content. CCTa association with the INM is mediated by a positively‐charged membrane‐binding M‐domain and a phosphorylated P‐domain containing 16 serine phospho‐sites. Mutation of all P‐domain serine residues to alanine (dephosphorylated mimic) enhanced association with nLDs in oleate‐treated U2OS cells, while mutation to glutamate (phosphorylated mimic) or mutation of M‐domain lysine residues ablated nLD association. However, it is unknown if phosphorylation regulation of CCTa association with the INM and nLDs involves specific serine residues or the net charge of the P‐domain. To address this question, we mutated 4 sets of 2‐3 serine residues in the P‐domain to alanine or aspartate. CCTa cDNAs expressing different combinations of these mutations were expressed in oleate‐treated U2OS cells and the frequency of nLD association was measured. The mutants were also expressed in CHO cells with a temperature‐sensitive CCTα isoform and INM translocation in response to oleate was determined. The greatest inhibitory effect on INM and nLD association was observed when all 4 sets of serine residues were mutated to aspartate and not by any one site, suggesting that net charge density of the P‐domain regulates affinity for the NE and nLD. Additionally, CCTa serine‐to‐alanine mutants expressed in U2OS cells were unstable compared to the corresponding aspartate mutants. We conclude that CCTa association with the INM and nLD is inhibited as overall P‐domain phosphorylation increases. In its active membrane‐associated, dephosphorylated state CCTa protein has decreased stability, a potently negative feedback mechanism to control PC synthesis. These results provide novel insight into how PC synthesis is regulated that will be essential for identifying kinases and phosphatases that act on CCTα.

Gut microbial β-glucuronidases regulate host luminal proteases and are depleted in irritable bowel syndrome.

Intestinal proteases mediate digestion and immune signaling, while increased gut proteolytic activity disrupts the intestinal barrier and generates visceral hypersensitivity, which in common in irritable bowel syndrome (IBS). However, the mechanisms controlling protease function are unclear. Here we show that members of the gut microbiota suppress intestinal proteolytic activity through production of unconjugated bilirubin. This occurs via microbial β-glucuronidase-mediated conversion of bilirubin conjugates. Metagenomic analysis of fecal samples from patients with post-infection IBS (n=52) revealed an altered gut microbiota composition, in particular a reduction in Alistipes taxa, and high gut proteolytic activity driven by specific host serine proteases compared to controls. Germ-free mice showed 10-fold higher proteolytic activity compared with conventional mice. Colonization with microbiota from high proteolytic activity IBS patients failed to suppress proteolytic activity in germ-free mice, but suppression of proteolytic activity was achieved with colonization using microbiota from healthy donors. High proteolytic activity mice had higher intestinal permeability, a higher relative abundance of Bacteroides and a reduction in Alistipes taxa compared with low proteolytic activity mice. High proteolytic activity IBS patients had lower fecal β-glucuronidase activity and end-products of bilirubin deconjugation. Mice treated with unconjugated bilirubin and β-glucuronidase overexpressing E. coli, which significantly reduced proteolytic activity, while inhibitors of microbial β-glucuronidases increased proteolytic activity. Together, these data define a disease-relevant mechanism of host-microbial interaction that maintains protease homeostasis in the gut.