The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells

AbstractSRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells

Patterns of expression, distribution and biological role of heterochromatin protein 1 (HP1) in human cell types

We have examined the distribution patterns of HP1α and HP1β under in vivo, ex vivo and in vitro conditions as well as in neoplastic samples. Consistent with a non-essential role in heterochromatin maintenance, both proteins are down-regulated during mammalian erythropoiesis and are diminished or undetectable in several types of terminally differentiated cells. Variant-specific patterns are observed in almost all human tissues examined. Yet, the most instructive example of HP1 plasticity is observed in the lymph nodes, where HP1α and HP1β exhibit regional patterns that are exactly complementary to one another. Furthermore, while HP1α exhibits a dispersed sub-nuclear distribution in the vast majority of peripheral blood lymphocytes, it coalesces into large heterochromatic foci upon stimulation with various mitogens and IL-2. The effect of proliferative signals on HP1α distribution is reproduced by co-culture of immortalized T- and B-cells and can be confirmed using cell cycle-specific markers. These complex patterns reveal an unexpected plasticity in HP1 variant expression and further suggest that the sub-nuclear distribution of HP1 proteins is regulated by humoral signals and microenvironmental cues. Furthermore, in GC-derived neoplasms the complementary distribution patterns of HP1α and HP1β cease to exist. The increase in the number of HP1β-positive neoplastic cells correlates with the clinical stage of the disease, an indication that the expression of this isoform may have some prognostic significance.Στην παρούσα μελέτη έγινε καταγραφή των προτύπων έκφρασης και κατανομής των HP1α και ΗΡ1β πρωτεϊνών σε in vivo, ex vivo και in vitro συνθήκες καθώς και σε νεοπλασματικά δείγματα. Σε συμφωνία με την άποψη ότι οι ΗΡ1 πρωτεΐνες δεν είναι απαραίτητες για τη διατήρηση της ετεροχρωματινικής υφής, παρατηρήθηκε ελάττωση των επιπέδων τους κατά τη διάρκεια της ερυθροποίησης καθώς και σε αρκετούς τύπους τελικώς διαφοροποιημένων κυττάρων. Παράλληλα, κύτταρο- και ιστο-ειδικά πρότυπα κατανομής παρατηρήθηκαν σχεδόν σε όλα τα είδη των ιστών που μελετήθηκαν. Το πιο αντιπροσωπευτικό παράδειγμα περιοχικής κατανομής των HP1α και ΗΡ1β καταγράφηκε στους λεμφαδένες οπού οι δυο ΗΡ1 ισόμορφες, στην Β-εξαρτωμένη ζώνη, παρουσιάζουν συμπληρωματικό πρότυπο εντόπισης. Επιπλέον, στα περιφερικά λεμφοκύτταρα η HP1α παρουσιάζει εναλλακτικά πρότυπα κατανομής (διάχυτο ή εστιακό φαινότυπο) που εξαρτώνται από το μικρο-περιβάλλον (παρουσία μιτογόνων ή κυτοκινών). Στα νεοπλάσματα του βλαστικού κέντρου η εντόπιση των HP1α και ΗΡ1β πρωτεϊνών παύει να είναι «συμπληρωματική». Η αύξηση του αριθμού των HP1β-θετικών νεοπλασματικών κυττάρων συσχετίζεται με τα όψιμα στάδια της νόσου και αποτελεί ένδειξη ότι η έκφραση της ισομορφής αυτής μπορεί να έχει διαγνωστική αξία

The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells.

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Objective: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. Methods: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). Results: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. Conclusions: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research. Keywords: Islets, Mitochondria, Respirometry, Glucos

Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis

Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.United States. National Institutes of Health (R01DK108056)United States. National Institutes of Health (R24DK090963

ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV.

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis.

BAT-controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet-induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold-stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1-cre (BAT-Mfn2-KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold-stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender-specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP-synthesizing fat oxidation, whereas in BAT from males, complex I-driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT-Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole-body cold-stimulated thermogenesis

Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels

Fasting hyperinsulinemia precedes the development of type 2 diabetes. However, it is unclear whether fasting insulin hypersecretion is a primary driver of insulin resistance or a consequence of the progressive increase in fasting glycemia induced by insulin resistance in the prediabetic state. Herein, we have discovered a mechanism that specifically regulates non-glucose-stimulated insulin secretion (NGSIS) in pancreatic islets that is activated by nonesterified free fatty acids, the major fuel used by β-cells during fasting. We show that the mitochondrial permeability transition pore regulator cyclophilin D (CypD) promotes NGSIS, but not glucose-stimulated insulin secretion, by increasing mitochondrial proton leak. Islets from prediabetic obese mice show significantly higher CypD-dependent proton leak and NGSIS compared with lean mice. Proton leak-mediated NGSIS is conserved in human islets and is stimulated by exposure to nonesterified free fatty acids at concentrations observed in obese subjects. Mechanistically, proton leak activates islet NGSIS independently of mitochondrial ATP synthesis but ultimately requires closure of the KATP channel. In summary, we have described a novel nonesterified free fatty acid-stimulated pathway that selectively drives pancreatic islet NGSIS, which may be therapeutically exploited as an alternative way to halt fasting hyperinsulinemia and the progression of type 2 diabetes

Titration of cell number per well for bioenergetics measurements in human PBMCs and CD4⁺ T cells.

<p>(A) Mitochondrial stress test seahorse profiles. (B). The relationship between the maximal respiration vs cell number is linear. (C) Ratio of the maximal respiration to the basal respiration is plotted against cell number. Resting PBMCs (N = 4), Activated PBMCs (N = 3), Resting T cells (N = 2), Activated T cells (N = 3).</p