Cells — Open Access Journal

The Notch family comprises a group of four ligand-dependent receptors that control evolutionarily conserved developmental and homeostatic processes and transmit signals to the microenvironment. NOTCH undergoes remodeling, maturation, and trafficking in a series of post-translational events, including glycosylation, ubiquitination, and endocytosis. The regulatory modifications occurring in the endoplasmic reticulum/Golgi precede the intramembrane γ-secretase proteolysis and the transfer of active NOTCH to the nucleus. Hence, NOTCH proteins coexist in different subcellular compartments and undergo continuous relocation. Various factors, including ion concentration, enzymatic activity, and co-regulatory elements control Notch trafficking. Interfering with these regulatory mechanisms represents an innovative therapeutic way to bar oncogenic Notch signaling. In this review, we briefly summarize the role of Notch signaling in cancer and describe the protein modifications required for NOTCH to relocate across different subcellular compartments. We focus on the functional relationship between these modifications and the corresponding therapeutic options, and our findings could support the development of trafficking modulators as a potential alternative to the well-known γ-secretase inhibitors. Full article

Estradiol, testosterone and other steroid hormones inhibit cytochrome c oxidase (CcO) purified from bovine heart. The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of K_i vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent. Binding of estradiol, but not of testosterone, brings about spectral shift of the oxidized CcO consistent with an effect on heme a₃³⁺. We presume that the hormones bind to CcO at the bile acid binding site described by Ferguson-Miller and collaborators. Estradiol is shown to inhibit intraprotein electron transfer between hemes a and a_3. Notably, neither estradiol nor testosterone suppresses the peroxidase activity of CcO. Such a specific mode of action indicates that inhibition of CcO activity by the hormones is associated with impairing proton transfer via the K-proton channel. Full article

Hyaluronic acid (HA) is a key component of the extracellular matrix of the lungs. A unique attribute of HA is its water-retaining properties, so HA has a major role in the regulation of fluid balance in the lung interstitium. Hyaluronic acid has been widely used in the treatment of eyes, ears, joints and skin disorders, but in the last years, it has been also proposed in the treatment of certain lung diseases, including airway diseases, due to its anti-inflammatory and water-binding capacities. Hyaluronic acid aerosol decreases the severity of elastase-induced emphysema in murine models, prevents bronchoconstriction in asthmatics and improves some functional parameters in chronic obstructive pulmonary disease (COPD) patients. Due to the protection of HA against bronchoconstriction and its hydration properties, inhaled HA would increase the volume of airway surface liquid, resulting in mucus hydration, increased mucous transport and less mucous plugging of the airways. In addition, it has been seen in human studies that the treatment with nebulised HA improves the tolerability of nebulised hypertonic saline (even at 6% or 7% of concentration), which has been demonstrated to be an effective treatment in bronchial secretion management in patients with cystic fibrosis and bronchiectasis. Our objective is to review the role of HA treatment in the management of chronic airway diseases. Full article

The increased interest in organoid research in recent years has contributed to an improved understanding of diseases that are currently untreatable. Various organoids, including kidney, brain, retina, liver, and spinal cord, have been successfully developed and serve as potential sources for regenerative medicine studies. However, the application of organoids has been limited by their lack of tissue components such as nerve and blood vessels that are essential to organ physiology. In this study, we used three-dimensional co-culture methods to develop colonic organoids that contained enteric nerves and blood vessels. The development of enteric nerves and blood vessels was confirmed phenotypically and genetically by the use of immunofluorescent staining and Western blotting. Colonic organoids that contain essential tissue components could serve as a useful model for the study of colon diseases and help to overcome current bottlenecks in colon disease research. Full article

The number of diabetic patients grows constantly worldwide. Many patients suffer simultaneously from diabetes mellitus type 2 (T2DM) and intervertebral disc disease (IVDD), suggesting a strong link between T2DM and IVDD. T2DM rodent models provide versatile tools to study this interrelation. We hypothesized that the previously achieved studies in rodents approved it. Performing a search in the publicly available electronic databases according to our inclusion (e.g., experimental study with clearly outlined methods investigating IVDD in diabetic rodent models) and exclusion (e.g., non-experimental) criteria, we included 23 studies from 1992 to 2020 analyzing different aspects of IVDD in diabetic rodents, such as on pathogenesis (e.g., effects of hyperglycemia on IVD cells, sirtuin (SIRT)1/p53 axis in the interrelation between T2DM and IVDD), risk factors (e.g., high content of advanced glycation end-products (AGEs) in modern diets), therapeutical approaches (e.g., insulin-like growth factor (IGF-I)), and prophylaxis. Regarding their quality, 12 studies were classified as high, six as moderate, and five as low. One strong, 18 moderate, and three mild evidences of the link between DM and IVDD in rodents were found, while only one study has not approved this link. We concluded that T2DM has a devastating effect on IVD, particularly in advanced cases, which needs to be further evaluated. Full article

The physiological fate of cells that die by apoptosis is their prompt and efficient removal by efferocytosis. During these processes, apoptotic cells release intracellular constituents that include purine nucleotides, lysophosphatidylcholine (LPC), and Sphingosine-1-phosphate (S1P) that induce migration and chemo-attraction of phagocytes as well as mitogens and extracellular membrane-bound vesicles that contribute to apoptosis-induced compensatory proliferation and alteration of the extracellular matrix and the vascular network. Additionally, during efferocytosis, phagocytic cells produce a number of anti-inflammatory and resolving factors, and, together with apoptotic cells, efferocytic events have a homeostatic function that regulates tissue repair. These homeostatic functions are dysregulated in cancers, where, aforementioned events, if not properly controlled, can lead to cancer progression and immune escape. Here, we summarize evidence that apoptosis and efferocytosis are exploited in cancer, as well as discuss current translation and clinical efforts to harness signals from dying cells into therapeutic strategies. Full article

COVID-19, caused by SARS-CoV-2 virus, emerged as a pandemic disease posing a severe threat to global health. To date, sporadic studies have demonstrated that innate immune mechanisms, specifically neutrophilia, NETosis, and neutrophil-associated cytokine responses, are involved in COVID-19 pathogenesis; however, our understanding of the exact nature of this aspect of host–pathogen interaction is limited. Here, we present a detailed dissection of the features and functional profiles of neutrophils, dendritic cells, and monocytes in COVID-19. We portray the crucial role of neutrophils as drivers of hyperinflammation associated with COVID-19 disease via the shift towards their immature forms, enhanced degranulation, cytokine production, and augmented interferon responses. We demonstrate the impaired functionality of COVID-19 dendritic cells and monocytes, particularly their low expression of maturation markers, increased PD-L1 levels, and their inability to upregulate phenotype upon stimulation. In summary, our work highlights important data that prompt further research, as therapeutic targeting of neutrophils and their associated products may hold the potential to reduce the severity of COVID-19. Full article

Cytokines are important contributors to immune responses against microbial and environmental threats and are of particular importance at epithelial barriers. These interfaces are continuously exposed to external factors and thus require immune components to both protect the host from pathogen invasion and to regulate overt inflammation. Recently, substantial efforts have been devoted to understanding how cytokines act on certain cells at barrier sites, and why the dysregulation of immune responses may lead to pathogenesis. In particular, the cytokine IL-22 is involved in preserving an intact epithelium, maintaining a balanced microbiota and a functioning defense system against external threats. However, a tight regulation of IL-22 is generally needed, since uncontrolled IL-22 production can lead to the progression of autoimmunity and cancer. Our aim in this review is to summarize novel findings on IL-22 and its interactions with specific microbial stimuli, and subsequently, to understand their contributions to the function of IL-22 and the clinical outcome. We particularly focus on understanding the detrimental effects of dysregulated control of IL-22 in certain disease contexts. Full article

Breast cancer is a heterogeneous disease that can be subdivided into unique molecular subtypes based on protein expression of the Estrogen Receptor, Progesterone Receptor, and/or the Human Epidermal Growth Factor Receptor 2. Therapeutic approaches are designed to inhibit these overexpressed receptors either by endocrine therapy, targeted therapies, or combinations with cytotoxic chemotherapy. However, a significant percentage of breast cancers are inherently resistant or acquire resistance to therapies, and mechanisms that promote resistance remain poorly understood. Notch signaling is an evolutionarily conserved signaling pathway that regulates cell fate, including survival and self-renewal of stem cells, proliferation, or differentiation. Deregulation of Notch signaling promotes resistance to targeted or cytotoxic therapies by enriching of a small population of resistant cells, referred to as breast cancer stem cells, within the bulk tumor; enhancing stem-like features during the process of de-differentiation of tumor cells; or promoting epithelial to mesenchymal transition. Preclinical studies have shown that targeting the Notch pathway can prevent or reverse resistance through reduction or elimination of breast cancer stem cells. However, Notch inhibitors have yet to be clinically approved for the treatment of breast cancer, mainly due to dose-limiting gastrointestinal toxicity. In this review, we discuss potential mechanisms of Notch-mediated resistance in breast cancer cells and breast cancer stem cells, and various methods of targeting Notch through γ-secretase inhibitors, Notch signaling biologics, or transcriptional inhibitors. We also discuss future plans for identification of novel Notch-targeted therapies, in order to reduce toxicity and improve outcomes for women with resistant breast cancer. Full article

Human α-synuclein expression in baker’s yeast reportedly induces mitochondria-dependent apoptosis. Surprisingly, we find that, under de-repressing conditions of the inducible MET25/GAL1 promoters, yeast cells expressing chromosomally-integrated copies of the human α-synuclein gene are not killed, but spontaneously form respiration-deficient rho-minus (ρ^-) petites. Although yeast cells can undergo cell death (apoptosis) from loss of mitochondrial function, they can also survive without functional mitochondria. Such cells are referred to as ρ⁰ or ρ^- petites. This study reports that minimal expression of human α-synuclein in yeast, from MET25/GAL1 promoter, gives rise to ρ^- petites. Interestingly, the full expression of α-synuclein, from the same promoters, in α-synuclein-triggered ρ^- petites and also in ρ⁰ petites (produced by treating ρ⁺ cells with the mutagen ethidium bromide) initiates apoptosis. The percentages of petites increase with increasing α-synuclein gene copy-number. ρ^- petites expressing α-synuclein from fully-induced MET25/GAL1 promoters exhibit increased ROS levels, loss of mitochondrial membrane potential, and nuclear DNA fragmentation, with increasing copies of α-synuclein. Our results indicate that, for the first time in yeast, α-synuclein-triggered apoptosis can occur independently of functional mitochondria. The observation that α-synuclein naturally forms petites and that they can undergo apoptosis may have important implications in understanding the pathogenesis of Parkinson’s disease. Full article

Signal transducer and activator of transcription 3 (STAT3) functions as a major molecular switch that plays an important role in the communication between cytokines and kinases. In this role, it regulates the transcription of genes involved in various biochemical processes, such as proliferation, migration, and metabolism of cancer cells. STAT3 undergoes diverse post-translational modifications, such as the oxidation of cysteine by oxidative stress, the acetylation of lysine, or the phosphorylation of serine/threonine. In particular, the redox modulation of critical cysteine residues present in the DNA-binding domain of STAT3 inhibits its DNA-binding activity, resulting in the inactivation of STAT3-mediated gene expression. Accumulating evidence supports that STAT3 is a key protein that acts as a mediator of metabolism and mitochondrial activity. In this review, we focus on the post-translational modifications of STAT3 by oxidative stress and how the modification of STAT3 regulates cell metabolism, particularly in the metabolic pathways in cancer cells. Full article

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G_q-coupled receptors and constitutively active Gα_q/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G_q/phospholipase C (PLC) signaling. Here, we further characterized G_q/11 regulation of SphK1. SphK1 translocation by the M₃ receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ₃, but accelerated by wild-type PLCβ₃ and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M₃ receptor-stimulated inositol phosphate production, suggesting competition at Gα_q. Embryonic fibroblasts from Gα_q/11 double-deficient mice were used to show that amino acids W263 and T257 of Gα_q, which interact directly with PLCβ₃ and p63RhoGEF, were important for bradykinin B₂ receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100–105 in human SphK1a), which resembles the Gα_q binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M₃ receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G_q/11. Full article

Podoplanin and CD44 are transmembrane glycoproteins involved in inflammation and cancer. In this paper, we report that podoplanin is coordinately expressed with the CD44 standard (CD44s) and variant (CD44v) isoforms in vivo—in hyperplastic skin after a pro-inflammatory stimulus with 12-O-tetradecanoylphorbol-13-acetate (TPA)—and in vitro—in cell lines representative of different stages of mouse-skin chemical carcinogenesis, as well as in human squamous carcinoma cell (SCC) lines. Moreover, we identify CD44v10 in the mouse-skin carcinogenesis model as the only CD44 variant isoform expressed in highly aggressive spindle carcinoma cell lines together with CD44s and podoplanin. We also characterized CD44v3-10, CD44v6-10 and CD44v8-10 as the major variant isoforms co-expressed with CD44s and podoplanin in human SCC cell lines. Immunofluorescence confocal microscopy experiments show that these CD44v isoforms colocalize with podoplanin at plasma membrane protrusions and cell–cell contacts of SCC cells, as previously reported for CD44s. Furthermore, CD44v isoforms colocalize with podoplanin in chemically induced mouse-skin SCCs in vivo. Co-immunoprecipitation experiments indicate that podoplanin physically binds to CD44v3-10, CD44v6-10 and CD44v8-10 isoforms, as well as to CD44s. Podoplanin–CD44 interaction is mediated by the transmembrane and cytosolic regions and is negatively modulated by glycosylation of the extracellular domain. These results point to a functional interplay of podoplanin with both CD44v and CD44s isoforms in SCCs and give insight into the regulation of the podoplanin–CD44 association. Full article

The concentration of circulating hematopoietic stem and progenitor cells has not been studied longitudinally. Here, we report that the proportions of Lin-CD34+38- hematopoietic multipotent cells (HMCs) and of Lin-CD34+CD38+ hematopoietic progenitors cells (HPCs) are highly variable between individuals but stable over long periods of time, in both healthy individuals and sickle cell disease (SCD) patients. This suggests that these proportions are regulated by genetic polymorphisms or by epigenetic mechanisms. We also report that in SCD patients treated with hydroxyurea, the proportions of circulating HMCs and HPCs show a strong positive and negative correlation with fetal hemoglobin (HbF) levels, respectively. Titration of 65 cytokines revealed that the plasma concentration of chemokines CCL2, CCL11, CCL17, CCL24, CCL27, and PDGF-BB were highly correlated with the proportion of HMCs and HPCs and that a subset of these cytokines were also correlated with HbF levels. A linear model based on four of these chemokines could explain 80% of the variability in the proportion of circulating HMCs between individuals. The proportion of circulating HMCs and HPCs and the concentration of these chemokines might therefore become useful biomarkers for HbF response to HU in SCD patients. Such markers might become increasingly clinically relevant, as alternative treatment modalities for SCD are becoming available. Full article

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of both upper and lower motor neurons. The disease presents a poor prognosis, and patients usually die 2–5 years after the onset of symptoms. The hallmark of this disease is the presence of phosphorylated and ubiquitinated aggregates containing trans-active response DNA-binding protein-43 (TDP-43) in the cytoplasm of motor neurons. TDP-43 pathology has been associated with multiple pathways in ALS, such as metabolic dysfunction found in patients and in in vivo models. Recently, it has been described as a “prion-like” protein, as studies have shown its propagation in cell culture from ALS brain extract or overexpressed TDP-43 in co-culture and conditioned medium, resulting in cytotoxicity. However, the cellular alterations that are associated with this cytotoxicity require further investigation. Here, we investigated the effects of conditioned medium from HEK293T (Human Embryonic Kidney 293T) cells overexpressing TDP-43 on cellular morphology, proliferation, death, and metabolism. Although we did not find evidence of TDP-43 propagation, we observed a toxicity of TDP-43-conditioned medium and altered metabolism. These results, therefore, suggest (1) that cells overexpressing TDP-43 produce an extracellular environment that can perturb other cells and (2) that TDP-43 propagation alone may not be the only potentially cytotoxic cell-to-cell mechanism. Full article

Murine fibroblasts deficient in mitochondria respiratory complexes III (CIII) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or Cox10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1–5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of CIII in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1α stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1α and increased CI levels in RISP and COX10 KO fibroblasts. Knockdown of HIF-1α during hypoxic conditions abrogated the beneficial effect of hypoxia on the stability/assembly of CI. These findings demonstrate that oxygen and HIF-1α regulate the assembly of respiratory complexes. Full article

Alzheimer’s disease (AD) is an age-related detrimental dementia. Amyloid beta peptides (Aβ) play a crucial role in the pathology of AD. In familial AD, Aβ are generated from the full-length amyloid beta precursor protein (APP) via dysregulated proteolytic processing; however, in the case of sporadic AD, the mechanism of Aβ biogenesis remains elusive. circRNAs are a class of transcripts preferentially expressed in brain. We identified a circRNA harboring the Aβ-coding region of the APP gene termed circAβ-a. This circular RNA was detected in the brains of AD patients and non-dementia controls. With the aid of our recently established approach for analysis of circRNA functions, we demonstrated that circAβ-a is efficiently translated into a novel Aβ-containing Aβ175 polypeptide (19.2 KDa) in both cultured cells and human brain. Furthermore, Aβ175 was shown to be processed into Aβ peptides—a hallmark of AD. In summary, our analysis revealed an alternative pathway of Aβ biogenesis. Consequently, circAβ-a and its corresponding translation product could potentially represent novel therapeutic targets for AD treatment. Importantly, our data point to yet another evolutionary route for potentially increasing proteome complexity by generating additional polypeptide variants using back-splicing of primary transcripts that yield circular RNA templates. Full article

The small nucleolar RNA snR30 (U17 in humans) plays a unique role during ribosome synthesis. Unlike most members of the H/ACA class of guide RNAs, the small nucleolar ribonucleoprotein (snoRNP) complex assembled on snR30 does not direct pseudouridylation of ribosomal RNA (rRNA), but instead snR30 is critical for 18S rRNA processing during formation of the small subunit (SSU) of the ribosome. Specifically, snR30 is essential for three pre-rRNA cleavages at the A₀/01, A₁/1, and A₂/2a sites in yeast and humans, respectively. Accordingly, snR30 is the only essential H/ACA guide RNA in yeast. Here, we summarize our current knowledge about the interactions and functions of snR30, discuss what remains to be elucidated, and present two non-exclusive hypotheses on the possible molecular function of snR30 during ribosome biogenesis. First, snR30 might be responsible for recruiting other proteins including endonucleases to the SSU processome. Second, snR30 may contribute to the refolding of pre-rRNA into a required conformation that serves as a checkpoint during ribosome biogenesis facilitating pre-rRNA cleavage. In both scenarios, the snR30 snoRNP may have scaffolding and RNA chaperoning activity. In conclusion, the snR30 snoRNP is a crucial player with an unknown molecular mechanism during ribosome synthesis, posing many interesting future research questions. Full article

Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified Mapk14 (p38) as a potential target to increase HSPC engraftment. Knockout of p38 prior to transplantation was sufficient to induce a selective advantage. Inhibition of p38 increased expression of the HSC homing factor CXCR4 and reduced apoptosis and proliferation in HSPCs. For potential clinical translation, treatment of IL1B-challenged or X-CGD HSPCs with a p38 inhibitor led to a 1.5-fold increase of donor cell engraftment. In summary, our findings demonstrate that p38 may serve as a potential druggable target to restore engraftment of HSPCs in the context of X-CGD gene therapy. Full article