Post-translational Mechanisms Research Articles

Regulation of cannabinoid and opioid receptor levels by endogenous and pharmacological chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of posttranslational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), delta opioid receptor (DOR), and CB1R-DOR interacting complexes. Focussing on endogenous protein chaperones namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared to other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of Cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. Significance Statement This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of CB1R, DOR, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

RNF149 Destabilizes IFNGR1 in Macrophages to Favor Postinfarction Cardiac Repair.

Macrophage-driven inflammation critically involves in cardiac injury and repair following myocardial infarction (MI). However, the intrinsic mechanisms that halt the immune response of macrophages, which is critical to preserve homeostasis and effective infarct repair, remain to be fully defined. Here, we aimed to determine the ubiquitination-mediated regulatory effects on averting exaggerated inflammatory responses in cardiac macrophages. We used transcriptome analysis of mouse cardiac macrophages and bone marrow-derived macrophages to identify the E3 ubiquitin ligase RNF149 (ring finger protein 149) as a modulator of macrophage response to MI. Employing loss-of-function methodologies, bone marrow transplantation approaches, and adenovirus-mediated RNF149 overexpression in macrophages, we elucidated the functional role of RNF149 in MI. We explored the underlying mechanisms through flow cytometry, transcriptome analysis, immunoprecipitation/mass spectrometry analysis, and functional experiments. RNF149 expression was measured in the cardiac tissues of patients with acute MI and healthy controls. RNF149 was highly expressed in murine and human cardiac macrophages at the early phase of MI. Knockout of RNF149, transplantation of Rnf149-/- bone marrow, and bone marrow macrophage-specific RNF149-knockdown markedly exacerbated cardiac dysfunction in murine MI models. Conversely, overexpression of RNF149 in macrophages attenuated the ischemia-induced decline in cardiac contractile function. RNF149 deletion increased infiltration of proinflammatory monocytes/macrophages, accompanied by a hastened decline in reparative subsets, leading to aggravation of myocardial apoptosis and impairment of infarct healing. Our data revealed that RNF149 in infiltrated macrophages restricted inflammation by promoting ubiquitylation-dependent proteasomal degradation of IFNGR1 (interferon gamma receptor 1). Loss of IFNGR1 rescued deleterious effects of RNF149 deficiency on MI. We further demonstrated that STAT1 (signal transducer and activator of transcription 1) activation induced Rnf149 transcription, which, in turn, destabilized the IFNGR1 protein to counteract type-II IFN (interferon) signaling, creating a feedback control mechanism to fine-tune macrophage-driven inflammation. These findings highlight the significance of RNF149 as a molecular brake on macrophage response to MI and uncover a macrophage-intrinsic posttranslational mechanism essential for maintaining immune homeostasis and facilitating cardiac repair following MI.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.

TDP43 autoregulation gives rise to shortened isoforms that are tightly controlled by both transcriptional and post-translational mechanisms.

The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.

Molecular Determinants of Sensitivity to Polatuzumab Vedotin in Diffuse Large B-Cell Lymphoma.

Polatuzumab vedotin (Pola-V) is an antibody-drug conjugate directed to the CD79B subunit of the B-cell receptor (BCR). When combined with conventional immunochemotherapy, Pola-V improves outcomes in diffuse large B-cell lymphoma (DLBCL). To identify determinants of Pola-V sensitivity, we used CRISPR-Cas9 screening for genes that modulated Pola-V toxicity for lymphomas or the surface expression of its target, CD79B. Our results reveal the striking impact of CD79B glycosylation on Pola-V epitope availability on the lymphoma cell surface and on Pola-V toxicity. Genetic, pharmacological, and enzymatic approaches that remove sialic acid from N-linked glycans enhanced lymphoma killing by Pola-V. Pola-V toxicity was also modulated by KLHL6, an E3 ubiquitin ligase that is recurrently inactivated in germinal center derived lymphomas. We reveal how KLHL6 targets CD79B for degradation in normal and malignant germinal center B cells, thereby determining expression of the surface BCR complex. Our findings suggest precision medicine strategies to optimize Pola-V as a lymphoma therapeutic. Significance: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, they reveal a novel post-translational mechanism used by normal and malignant germinal center B cells to regulate expression of the BCR. See related commentary by Leveille, p. 1577 See related article by Meriranta et al.

Targeting a disintegrin and metalloprotease (ADAM) 17-CD122 axis enhances CD8+ T cell effector differentiation and anti-tumor immunity

CD8+ T cell immune responses are regulated by multi-layer networks, while the post-translational regulation remains largely unknown. Transmembrane ectodomain shedding is an important post-translational process orchestrating receptor expression and signal transduction through proteolytic cleavage of membrane proteins. Here, by targeting the sheddase A Disintegrin and Metalloprotease (ADAM)17, we defined a post-translational regulatory mechanism mediated by the ectodomain shedding in CD8+ T cells. Transcriptomic and proteomic analysis revealed the involvement of post-translational regulation in CD8+ T cells. T cell-specific deletion of ADAM17 led to a dramatic increase in effector CD8+ T cell differentiation and enhanced cytolytic effects to eliminate pathogens and tumors. Mechanistically, ADAM17 regulated CD8+ T cells through cleavage of membrane CD122. ADAM17 inhibition led to elevated CD122 expression and enhanced response to IL-2 and IL-15 stimulation in both mouse and human CD8+ T cells. Intriguingly, inhibition of ADAM17 in CD8+ T cells improved the efficacy of chimeric antigen receptor (CAR) T cells in solid tumors. Our findings reveal a critical post-translational regulation in CD8+ T cells, providing a potential therapeutic strategy of targeting ADAM17 for effective anti-tumor immunity.

Expression and localization of anti-Müllerian hormone and its receptors in bovine corpus luteum

Although anti-Müllerian hormone (AMH) is involved in the regulation of granulosa cell function in female animals, its role in tissues other than ovarian follicles remains poorly understood. It has also been suggested that cows with high circulating AMH concentrations have increased fertility; however, the mechanism has not been elucidated. This study was conducted to identify the presence of the AMH-signaling system and its target cells in the bovine corpus luteum formed from an ovulated follicle. Immunoblotting revealed that the proteolytically cleaved C-terminal region in AMH (AMHC), a biologically active peptide, was present in trace amounts in the early corpus luteum and significantly increased during the mid to regressed stages. AMHC and cleaved N-terminal region (AMHN) in AMH generate a noncovalent isoform that improves the activity of AMH signaling. An immunohistochemical analysis revealed that AMHC, AMHN, and type II AMH receptor (AMHR2) were localized to luteal cells during the entire estrous cycle. AMH in the corpus luteum seemed to be newly synthesized since AMH expression was detected. These findings suggest that AMH signaling is involved in the regulation of luteal cell function through an autocrine and post-translational processing mechanism. The level of AMHR2 and mRNA expression of AMHR2 and type I AMH receptors (activin-like kinase 2, 3, and 6) were highest in the mid stage. Thus, AMH signaling in the corpus luteum may also be regulated by changes in the receptor levels. Since the transforming growth factor-beta superfamily, to which AMH belongs, is a multifunctional polypeptide growth factor, further studies are needed to evaluate whether AMH signaling has a role in facilitating or inhibiting luteal cell functions.

Posttranslational regulation of photosynthetic activity via the TOR kinase in plants.

Chloroplasts are the powerhouse of the plant cell, and their activity must be matched to plant growth to avoid photooxidative damage. We have identified a posttranslational mechanism linking the eukaryotic target of rapamycin (TOR) kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origins that regulates chloroplast activity and photosynthesis in particular. We find that RelA SpoT homolog 3 (RSH3), a nuclear-encoded enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific amino-terminal region which is phosphorylated in a TOR-dependent manner. Down-regulating TOR activity causes a rapid increase in ppGpp synthesis in RSH3 overexpressors and reduces photosynthetic capacity in an RSH-dependent manner in wild-type plants. The TOR-RSH3 signaling axis therefore regulates the equilibrium between chloroplast activity and plant growth, setting a precedent for the regulation of organellar function by TOR.

Multiplexed Profiling of CDK Kinase Activities in Tumor Biopsies with Fluorescent Peptide Biosensors.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

PARP14 is regulated by the PARP9/DTX3L complex and promotes interferon γ-induced ADP-ribosylation

Protein ADP-ribosylation plays important but ill-defined roles in antiviral signalling cascades such as the interferon response. Several viruses of clinical interest, including coronaviruses, express hydrolases that reverse ADP-ribosylation catalysed by host enzymes, suggesting an important role for this modification in host-pathogen interactions. However, which ADP-ribosyltransferases mediate host ADP-ribosylation, what proteins and pathways they target and how these modifications affect viral infection and pathogenesis is currently unclear. Here we show that host ADP-ribosyltransferase activity induced by IFNγ signalling depends on PARP14 catalytic activity and that the PARP9/DTX3L complex is required to uphold PARP14 protein levels via post-translational mechanisms. Both the PARP9/DTX3L complex and PARP14 localise to IFNγ-induced cytoplasmic inclusions containing ADP-ribosylated proteins, and both PARP14 itself and DTX3L are likely targets of PARP14 ADP-ribosylation. We provide evidence that these modifications are hydrolysed by the SARS-CoV-2 Nsp3 macrodomain, shedding light on the intricate cross-regulation between IFN-induced ADP-ribosyltransferases and the potential roles of the coronavirus macrodomain in counteracting their activity.

Ubiquitin ligase RFWD2 promotes dendritic spine and synapse formation by activating the ERK/PEA3/c-Jun pathway in rat cerebral cortical neurons

The regulation of protein degradation through the ubiquitin–proteasome system is essential for normal brain development, axon growth, synaptic growth and plasticity. The E3 ubiquitin ligase RFWD2 plays a key role in the onset and development of neurological diseases, including the pathogenesis of Alzheimer's disease (AD), but the mechanisms controlling the homeostasis of neuronal synaptic proteins are still poorly understood. Here, we showed that the expression level of RFWD2 gradually decreased with the age of the rats and was negatively correlated with the development of cerebral cortical neurons and dendrites in vivo. RFWD2 was shown to localize to presynaptic terminals and some postsynaptic sides of both excitatory synapses and inhibitory synapses via colocalization with neuronal synaptic proteins (SYN, PSD95, Vglut1 and GAD67). Overexpression of RFWD2 promoted dendrite development and dendritic spine formation and markedly decreased the expression of synaptophysin and PSD95 by reducing the expression of ETV1, ETV4, ETV5 and c-JUN in vitro. Furthermore, the whole-cell membrane slice clamp results showed that RFWD2 overexpression resulted in greater membrane capacitance in neuronal cells, inadequate cell repolarization, and a longer time course for neurons to emit action potentials with decreased excitability. RFWD2 regulates dendritic development and plasticity, dendritic spine formation and synaptic function in rat cerebral cortex neurons by activating the ERK/PEA3/c-Jun pathway via a posttranslational regulatory mechanism and can be used as an efficient treatment target for neurological diseases.

Posttranslational regulatory mechanism of PD-L1 in cancers and associated opportunities for novel small-molecule therapeutics.

Despite the tremendous progress in cancer research over the past few decades, effective therapeutic strategies are still urgently needed. Accumulating evidence suggests that immune checkpoints are the cause of tumor immune escape. PD-1/PD-L1 are among them. Posttranslational modification is the most critical step for protein function, and the regulation of PD-L1 by small molecules through posttranslational modification is highly valuable. In this review, we discuss the mechanisms of tumor cell immune escape and several posttranslational modifications associated with PD-L1 and describe examples in which small molecules can regulate PD-L1 through posttranslational modifications. Herein, we propose that the use of small molecule compounds that act by inhibiting PD-L1 through posttranslational modifications is a promising therapeutic approach with the potential to improve clinical outcomes for cancer patients.

Predictive modeling for ubiquitin proteins through advanced machine learning technique

Ubiquitination is an essential post-translational modification mechanism involving the ubiquitin protein's bonding to a substrate protein. It is crucial in a variety of physiological activities including cell survival and differentiation, and innate and adaptive immunity. Any alteration in the ubiquitin system leads to the development of various human diseases. Numerous researches show the highly reversibility and dynamic of ubiquitin system, making the experimental identification quite difficult. To solve this issue, this article develops a model using a machine learning approach, tending to improve the ubiquitin protein prediction precisely. We deeply investigate the ubiquitination data that is proceed through different features extraction methods, followed by the classification. The evaluation and assessment are conducted considering Jackknife tests and 10-fold cross-validation. The proposed method demonstrated the remarkable performance in terms of 100 %, 99.88 %, and 99.84 % accuracy on Dataset-I, Dataset-II, and Dataset-III, respectively. Using Jackknife test, the method achieves 100 %, 99.91 %, and 99.99 % for Dataset-I, Dataset-II and Dataset-III, respectively. This analysis concludes that the proposed method outperformed the state-of-the-arts to identify the ubiquitination sites and helpful in the development of current clinical therapies. The source code and datasets will be made available at Github.

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite- as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

MEG3 in hematologic malignancies: from the role of disease biomarker to therapeutic target.

Maternally expressed gene 3 ( MEG3 ) is a noncoding RNA that is known as a tumor suppressor in solid cancers. Recently, a line of studies has emphasized its potential role in hematological malignancies in terms of tumorigenesis, metastasis, and drug resistance. Similar to solid cancers, MEG3 can regulate various cancer hallmarks via sponging miRNA, transcriptional, or posttranslational regulation mechanisms, but may regulate different key elements. In contrast with solid cancers, in some subtypes of leukemia, MEG3 has been found to be upregulated and oncogenic. In this review, we systematically describe the role and underlying mechanisms of MEG3 in multiple types of hematological malignancies. Particularly, we highlight the role of MEG3 in drug resistance and as a novel therapeutic target.

New insights into the regulation of bile acids synthesis during the early stages of liver regeneration: A human and experimental study

Background and aims: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol.Methods: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot.Results: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet.Conclusions: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.