High Rate Of Protein Synthesis Research Articles

Impaired Long-Chain Fatty Acid Metabolism in Carnitine Palmitoyltransferase II Knockout Mouse Leads to Chronic Pancreatitis

Background: To support high rates of protein synthesis, pancreatic acinar cells must generate large quantities of ATP. Mitochondrial dysfunction and impaired energy metabolism play a significant role in the development of pancreatitis. Patients with elevated plasma triglycerides are at increased risk for development of severe acute pancreatitis. Carnitine palmitoyltransferase II (CPT2) is required for importing and thus metabolizing long-chain fatty acids (LCFAs) in mitochondria. To inhibit LCFA metabolism, we utilized the CPT2acinarKO mouse to probe the consequences of impaired LCFA import on pancreatic function. Hypothesis: Loss of CPT2 causes an accumulation of long-chain acylcarnitines and disruption of the tissue acylcarnitine/free carnitine ratio which is thought to alter cellular function. Given acinar cells’ high metabolic requirement, impaired LCFA utilization would reduce energy production, secretory function, and alter pancreatic lipid composition. Methods: We generated mice lacking CPT2 specifically in acinar cells (CPT2acinarKO) by crossing tamoxifen-inducible, Elastase-Cre mice with CPT2 floxed mice. Mice were fed a high fat (60% kcal fat), low fat (10% kcal fat), or chow (14% kcal fat) diet for 12 weeks. Analysis of fat accumulation, edema, and fibrosis was performed with H&E and picrosirius staining. Mass Spec. lipid profiling was performed in pancreas and serum. Acinar cells were isolated from wildtype, CPT2 floxed, and CPT2acinarKO mice to assess secretory function, ATP levels, and necrosis via lactate dehydrogenase (LDH) release. Results: Histologic examination revealed CPT2acinarKO mice on all three diets displayed pancreatic fat accumulation. Strikingly, CPT2acinarKO mice fed a chow or low fat diet developed chronic pancreatitis with a 25% loss in pancreatic weight, immune cell infiltration, edema, and fibrosis. As anticipated, long-chain acylcarnitines were significantly elevated in CPT2acinarKO pancreas. Assessment of secretion in 2 weeks post tamoxifen cells revealed significantly reduced maximal (10 pM) and supramaximal (1 nM) cholecystokinin (CCK) stimulated amylase release as a percent of total cellular amylase. Acini isolated at 4 weeks post tamoxifen revealed significantly increased basal secretion and reduced maximal and supramaximal CCK stimulated amylase release. When normalized to cellular DNA, secretion was similar between CPT2acinarKO and CPT2 floxed acinar cells indicating CPT2acinarKO cells had reduced amylase levels. Wildtype acinar cells treated with unsaturated and saturated free fatty acid concentrations as low as 100 μM for 3 hours caused significant necrosis. CPT2acinarKO pancreas had significantly decreased ATP levels indicative of mitochondrial dysfunction. Conclusion: Loss of CPT2 in the pancreatic acinar cells impairs LCFA import, reduces ATP levels, disrupts the secretory pathway, promoting chronic pancreatitis. In contrast to other CPT2 knockout models, pancreatitis occurs independent of high fat feeding in CPT2acinarKO mice. Further investigation is needed to identify metabolic alterations leading to acinar damage and mitochondrial dysfunction following the loss of CPT2. UW-Madison Comprehensive Diabetes Center Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macula densa mTORC1 is an important regulator of cellular and aging-dependent kidney functions

Macula densa (MD) cells are major regulators of nephron, kidney, and whole body functions, including the control of renal blood flow, glomerular filtration rate (GFR), renin release and thus the maintenance of body fluid and electrolyte balance. Our recent studies identified the high rate of MD protein synthesis regulated by the mTORC1 complex of the mechanistic target of rapamycin (mTOR) as a novel component of MD cell and glomerular hemodynamic regulatory functions. Raptor is an essential player in mTORC1’s key roles to regulate cell energy homeostasis, growth, protein synthesis, mitochondrial function, apoptosis, and autophagy via phosphorylation of substrates including ribosomal protein S6 kinase (S6K) and eukaryotic translation-initiation factor 4E-binding proteins (4E-BPs). mTORC1 signaling controls the aging process, and deficient mTORC1 activity extends health and lifespan. Therefore, we hypothesized that MD cell-specific mTOR loss of function (lof) prevents aging-related kidney function decline. The present study aimed to demonstrate the key importance of MD cell mTOR activity in renal physiology and aging by characterizing the phenotype of a newly generated genetic MD-mTOR lof mouse model. MD-mTOR lof mice were generated by crossing Nos1-CreERT2 and Raptor floxed mice. Tamoxifen induction (at 3 weeks of age) of Nos1-CreERT2+/- Raptor−/− mice resulted in conditional and inducible Raptor deletion in cells of the Nos1 lineage, which in the kidney is entirely specific to MD cells. Kidney structure/function phenotyping was performed using intravital multiphoton microscopy (MPM), the Medibeacon transdermal GFR measurement system, and tissue harvest, immunohistochemistry, and the OPP incorporation assay of protein synthesis. High and MD-specific Raptor, pS6K, and p4EBP1 immunofluorescence labeling was observed in WT kidney sections. The immunolabeling of these mTOR component and targets were absent specifically in MD cells in MD-mTOR lof mouse kidneys, validating the MD cell-specific genetic approach. The intensity of incorporated OPP labeling was highest in MD cells among all renal cell types in WT. In contrast, MD OPP labeling was significantly reduced in MD-mTOR lof mice indicating diminished, mTORC1-dependent global MD cell protein synthesis. Importantly, in 15-month old aged mice the GFR was significantly higher (preserved) in MD-mTOR lof (1794±197 uL/min/100g BW) compared to WT (1117±61) and MD-mTOR gof (929±61) mice (n=6-18 mice in each group). Altogether, these studies identified mTORC1 as an essential regulator of MD cell biology, and the importance of MD cells in aging-related kidney function decline. MD cells may be important new targets in future anti-aging therapies. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integration of physiological and gene expression analyses to reveal biomarkers for protein dynamic mechanisms regulating higher growth and survival among larval oyster families (Crassostrea gigas)

The oyster Crassostrea gigas is a major, global aquaculture species. As with any domestically-farmed species, the characterization of breeding lines that yield desired phenotypes is of immense value. An understanding of the fundamental biological bases of such phenotypes is needed to enhance aquaculture production. The aim of our study was to investigate the mechanisms of protein metabolic dynamics and energy allocation in oyster larvae. A series of controlled crosses yielded full-sibling larval families that allowed for measurements of integrative physiological processes during development. Experimentally, phenotypic contrasts between larval families were assayed by measuring: (1) growth and survival, (2) utilization of energy reserves of lipid and protein, (3) rates of protein synthesis and turnover, (4) respiration rates, and (5) transcriptome gene expression. Initially, newly-formed 2-day-old veliger larvae from four different families had similar sizes and physiologies, as measured by respiration, protein synthesis, turnover and content, the amount of energy allocated to protein synthesis, and gene expression pattern. Upon feeding, notable phenotypic contrasts became evident in different families. The larval family with faster growth had higher rates of protein synthesis and allocated a higher percentage of available energy to that single process. Based on family-specific differences, a series of samples was selected for developmental time-course analysis of changes in RNA pools. Principal component analyses of family-specific differential gene expression, combined with measured biochemical and physiological processes, led to the identification of two ribosomal gene biomarkers for protein synthesis. Such biomarkers could be potentially valuable tools for assessing complex traits that regulate physiological state, leading to optimization of breeding programs for oyster aquaculture.

An approach to learn regulation to maximize growth and entropy production rates in metabolism

Elucidating cell regulation remains a challenging task due to the complexity of metabolism and the difficulty of experimental measurements. Here we present a method for prediction of cell regulation to maximize cell growth rate while maintaining the solvent capacity of the cell. Prediction is formulated as an optimization problem using a thermodynamic framework that can leverage experimental data. We develop a formulation and variable initialization procedure that allows for computing solutions of the optimization with an interior point method. The approach is applied to photoheterotrophic growth of Rhodospirilium rubrum using ethanol as a carbon source, which has applications to biosynthesis of ethylene production. Growth is captured as the rate of synthesis of amino acids into proteins, and synthesis of nucleotide triphoshaptes into RNA and DNA. The method predicts regulation that produces a high rate of protein and RNA synthesis while DNA synthesis is reduced close to zero in agreement with production of DNA being turned off for much of the cell cycle.

Vegan and Omnivorous High Protein Diets Support Comparable Daily Myofibrillar Protein Synthesis Rates and Skeletal Muscle Hypertrophy in Young Adults

BackgroundIt remains unclear whether non–animal-derived dietary protein sources (and therefore vegan diets) can support resistance training-induced skeletal muscle remodeling to the same extent as animal-derived protein sources. MethodsIn Phase 1, 16 healthy young adults (m = 8, f = 8; age: 23 ± 1 y; BMI: 23 ± 1 kg/m2) completed a 3-d dietary intervention (high protein, 1.8 g·kg bm−1·d−1) where protein was derived from omnivorous (OMNI1; n = 8) or exclusively non-animal (VEG1; n = 8) sources, alongside daily unilateral leg resistance exercise. Resting and exercised daily myofibrillar protein synthesis (MyoPS) rates were assessed using deuterium oxide. In Phase 2, 22 healthy young adults (m = 11, f = 11; age: 24 ± 1 y; BMI: 23 ± 0 kg/m2) completed a 10 wk, high-volume (5 d/wk), progressive resistance exercise program while consuming an omnivorous (OMNI2; n = 12) or non–animal-derived (VEG2; n = 10) high-protein diet (∼2 g·kg bm−1·d−1). Muscle fiber cross-sectional area (CSA), whole-body lean mass (via DXA), thigh muscle volume (via MRI), muscle strength, and muscle function were determined pre, after 2 and 5 wk, and postintervention. ObjectivesTo investigate whether a high-protein, mycoprotein-rich, non-animal-derived diet can support resistance training-induced skeletal muscle remodeling to the same extent as an isonitrogenous omnivorous diet. ResultsDaily MyoPS rates were ∼12% higher in the exercised than in the rested leg (2.46 ± 0.27%·d−1 compared with 2.20 ± 0.33%·d−1 and 2.62 ± 0.56%·d−1 compared with 2.36 ± 0.53%·d−1 in OMNI1 and VEG1, respectively; P < 0.001) and not different between groups (P > 0.05). Resistance training increased lean mass in both groups by a similar magnitude (OMNI2 2.6 ± 1.1 kg, VEG2 3.1 ± 2.5 kg; P > 0.05). Likewise, training comparably increased thigh muscle volume (OMNI2 8.3 ± 3.6%, VEG2 8.3 ± 4.1%; P > 0.05), and muscle fiber CSA (OMNI2 33 ± 24%, VEG2 32 ± 48%; P > 0.05). Both groups increased strength (1 repetition maximum) of multiple muscle groups, to comparable degrees. ConclusionsOmnivorous and vegan diets can support comparable rested and exercised daily MyoPS rates in healthy young adults consuming a high-protein diet. This translates to similar skeletal muscle adaptive responses during prolonged high-volume resistance training, irrespective of dietary protein provenance.This trial was registered at clinicaltrials.gov as NCT03572127.

Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation

BackgroundProlonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss.ResultsWe used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD.ConclusionsWe provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

Protein Homeostasis Is Dynamically Regulated throughout Hematopoietic Stem Cell Ontogeny

Hematopoietic stem cells (HSCs) are required for establishing hematopoiesis during development and for maintaining lifelong regeneration of blood cells. As demands for blood cell production change throughout ontogeny, there is a commensurate change in HSC biology. Fetal, neonatal, and adult HSCs exhibit differences in gene expression, developmental potential, and self-renewal activity. Thus, fetal, neonatal, and adult HSCs are, at least functionally, distinct populations. We recently found that fetal HSCs exhibit substantially higher rates of protein synthesis as compared to adult HSCs, which depend on unusually low protein synthesis rates to limit the biogenesis of misfolded and unfolded proteins to preserve protein homeostasis (proteostasis) and fitness. This raised questions of whether fetal HSCs experience significant proteotoxic stress in vivo and how proteostasis is regulated throughout HSC development. Remarkably, we found that fetal HSCs restrict accumulation of unfolded proteins to levels similar to those observed in adult HSCs, despite having nearly 7-fold higher protein synthesis rates. Surprisingly, however, we determined that unfolded protein abundance spikes dramatically within HSCs at birth. This transient burst of proteotoxic stress is followed by rapid restoration of proteostasis in neonates, as unfolded protein abundance declines precipitously, ultimately reaching unusually low levels by P14 before moderately rising again in adulthood. In contrast to the dynamic oscillation of unfolded protein abundance, protein synthesis rates progressively decline throughout HSC ontogeny. Fetal HSCs synthesize significantly more protein per hour than HSCs from newborn and postnatal mice, which in turn have nearly 3-fold and 2-fold higher protein synthesis rates than adult HSCs, respectively. Strikingly, however, postnatal declines in HSC protein synthesis occur gradually and non-uniformly. While fetal and adult HSCs have narrow distributions of protein synthesis rates, neonatal HSCs exhibit significant heterogeneity, suggesting an uncoordinated transition to achieving low protein synthesis rates and proteostasis in adulthood. Taken together, these findings indicate that HSC ontogeny is associated with rapid, dynamic, and robust changes in proteostasis activity, and that fetal, neonatal, and adult HSCs utilize distinct mechanisms to regulate proteostasis in vivo. The regulation of proteostasis is thus a key distinguishing feature of HSC ontogeny that could provide a framework to uncover how pathogenic changes disrupt hematopoietic development and function in an age-specific manner. Furthermore, harnessing developmental proteostasis mechanisms could open new avenues to restore HSC fitness in response to various stressors and blood disorders that emerge throughout life.

Periostin- and podoplanin-positive cancer-associated fibroblast subtypes cooperate to shape the inflamed tumor microenvironment in aggressive pancreatic adenocarcinoma.

Cancer-associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in-house and public, including single-cell analyses), genetically engineered mouse PDAC tissues, and patient-derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin-positive CAFs were significantly associated with shorter overall survival of patients. Periostin-positive CAFs were characterized by high proliferation and protein synthesis rates and low α-smooth muscle actin expression and were found in peri-/pre-tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin-positive CAFs were associated with immune-related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin-positive CAFs and podoplanin-positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin-positive CAFs identified an inflammatory CAF (iCAF)-like subset, whereas periostin-positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

MTORC1 regulates high levels of protein synthesis in retinal ganglion cells of adult mice

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.

Urocortin 2 promotes hypertrophy and enhances skeletal muscle function through cAMP and insulin/IGF-1 signaling pathways

ObjectiveAlthough it is well established that urocortin 2 (Ucn2), a peptide member of the corticotrophin releasing factor (CRF) family, and its specific corticotrophin-releasing factor 2 receptor (CRF2R) are highly expressed in skeletal muscle, the role of this peptide in the regulation of skeletal muscle mass and protein metabolism remains elusive. MethodsTo elucidate the mechanisms how Ucn2 directly controls protein metabolism in skeletal muscles of normal mice, we carried out genetic tools, physiological and molecular analyses of muscles in vivo and in vitro. ResultsHere, we demonstrated that Ucn2 overexpression activated cAMP signaling and promoted an expressive muscle hypertrophy associated with higher rates of protein synthesis and activation of Akt/mTOR and ERK1/2 signaling pathways. Furthermore, Ucn2 induced a decrease in mRNA levels of atrogin-1 and in autophagic flux inferred by an increase in the protein content of LC3-I, LC3-II and p62. Accordingly, Ucn2 reduced both the transcriptional activity of FoxO in vivo and the overall protein degradation in vitro through an inhibition of lysosomal proteolytic activity. In addition, we demonstrated that Ucn2 induced a fast-to-slow fiber type shift and improved fatigue muscle resistance, an effect that was completely blocked in muscles co-transfected with mitogen-activated protein kinase phosphatase 1 (MKP-1), but not with dominant-negative Akt mutant (Aktmt). ConclusionsThese data suggest that Ucn2 triggers an anabolic and anti-catabolic response in skeletal muscle of normal mice probably through the activation of cAMP cascade and participation of Akt and ERK1/2 signaling. These findings open new perspectives in the development of therapeutic strategies to cope with the loss of muscle mass.

Calreticulin expression and localization in relation to exchangeable Ca2+ during pollen development in Petunia

BackgroundPollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca2+). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development.ResultsUsing northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm.ConclusionOur present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development.

A new view of macula densa cell protein synthesis.

Macula densa (MD) cells, a chief sensory cell type in the nephron, are endowed with unique microanatomic features including a high density of protein synthetic organelles and secretory vesicles in basal cell processes ("maculapodia") that suggest a so far unknown high rate of MD protein synthesis. This study aimed to explore the rate and regulation of MD protein synthesis and their effects on glomerular function using novel transgenic mouse models, newly established fluorescence cell biology techniques, and intravital microscopy. Sox2-tdTomato kidney tissue sections and an O-propargyl puromycin incorporation-based fluorescence imaging assay showed that MD cells have the highest level of protein synthesis within the kidney cortex followed by intercalated cells and podocytes. Genetic gain of function of mammalian target of rapamycin (mTOR) signaling specifically in MD cells (in MD-mTORgof mice) or their physiological activation by low-salt diet resulted in further significant increases in the synthesis of MD proteins. Specifically, these included both classic and recently identified MD-specific proteins such as cyclooxygenase 2, microsomal prostaglandin E2 synthase 1, and pappalysin 2. Intravital imaging of the kidney using multiphoton microscopy showed significant increases in afferent and efferent arteriole and glomerular capillary diameters and blood flow in MD-mTORgof mice coupled with an elevated glomerular filtration rate. The presently identified high rate of MD protein synthesis that is regulated by mTOR signaling is a novel component of the physiological activation and glomerular hemodynamic regulatory functions of MD cells that remains to be fully characterized.NEW & NOTEWORTHY This study discovered the high rate of protein synthesis in macula densa (MD) cells by applying direct imaging techniques with single cell resolution. Physiological activation and mammalian target of rapamycin signaling played important regulatory roles in this process. This new feature is a novel component of the tubuloglomerular cross talk and glomerular hemodynamic regulatory functions of MD cells. Future work is needed to elucidate the nature and (patho)physiological role of the specific proteins synthesized by MD cells.

Distinct MiR-126 Expression Describes a Non-Genetic but Functional Intra-Tumoral Heterogeneity in Primary Human B-ALL and Can be Prospectively Used to Identify Aggressive Subclones in NSG Mice

Intra-tumor heterogeneity is a hallmark of B acute lymphoblastic leukemia (B-ALL) favoring dissociated treatment responses, clonal evolution and disease progression. Heterogeneity at the genetic level has been widely described, but prospective isolation of functionally relevant, rare blast subpopulation within the B-ALL bulk remains challenging. We have found that all human adult B-ALL we tested (n=11) contained up to 4 subpopulations differing in the expression level of a stem cell microRNA, miR-126, which regulates quiescence of normal hematopoietic and AML leukemic stem cells (Lechman et al., Cancer Cell 2016). Transducing primary human B-ALL with a lentiviral miR-126 reporter, we can detect and purify within a single disease distinct B-ALL subsets differing in miR-126 expression. These miR-126_variants do not correspond to distinct genetic subclones, since they harbor, when purified, the same immunoglobulin/TCR rearrangements and show similar variant allele frequencies as the ones found by whole exome sequencing in the disease bulk. Surprisingly, when we xenotransplanted purified miR-126_variants, they stably maintained their associated miR-126 activity throughout tertiary transplantation, suggesting that they are not hierarchically-related but represent distinct, non-genetically-coded subclones. We next transplanted miR-126_variants at limiting dilution to measure their leukemia-initiating cell (LIC) frequency. LIC frequency was similar in miR-126(high) and miR-126(low) subpopulations. However, the miR-126(high) subsets from a given disease (n=2 BCR-ABL1-rearranged B-ALL) had more aggressive growth kinetics than their miR-126(low) counterparts and were enriched during chemotherapy with vincristine and dexamethasone. We then performed RNA sequencing of miR-126_variants comparing the miR-126(high) to the miR-126(low) subpopulation from the same primary B-ALL (n=3 BCR-ABL1-rearranged B-ALL cases). Differentially regulated gene ontologies in miR-126(high) versus (low) subpopulations common to all 3 diseases include PI3K/AKT signaling (a well-known, previously validated miR-126 target pathway), metabolism, cell cycle, TP53 and cell-cell interactions. We started to functionally validate some of these pathways, focusing on metabolic activity. We measured the rate of protein synthesis in miR-126(high) and miR-126(low) subpopulations by OPP incorporation and confirmed a higher rate of protein synthesis in the miR-126(high) subset. Intriguingly, the transcriptional profile of naturally-occurring miR-126_variants in human B-ALL resembled a miR-126 withdrawal signature in an experimental, miR-126-addicted B-ALL mouse model (Nucera et al., Cancer Cell 2016), pointing to a pathogenetic relevance of miR-126 also in the human disease. More in-depth studies on clinical samples, including diagnosis-relapse pairs, are ongoing. In conclusion, miR-126 can be used to prospectively dissect B-ALL heterogeneity, and understanding its molecular targets may open up new treatment approaches directed to specific functional cell states relevant to B-ALL biology. DisclosuresCiceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.

Age-adjusted mortality from pancreatic cancer increased NINE-FOLD in japan from 1950 to 1995 – Was a low-protein quasi-vegan diet a key factor in their former low risk?

During the last half of the twentieth century, age-adjusted mortality from pancreatic cancer in Japan rose about nine-fold in both sexes. Well-characterized risk factors such as smoking, obesity/metabolic syndrome, and heavy alcohol use appear to explain only a modest part of this rise. It is proposed that a diet relatively low in protein, and particularly low in animal protein, was a key determinant of the low risk for pancreatic cancer in mid-century Japan. It is further proposed that pancreatic acinar cells, owing to their extraordinarily high rate of protein synthesis, are at high risk for ER stress; that such stress plays a fundamental role in the induction of most pancreatic cancers; and that low-protein diets help to offset such stress by modulating activities of the kinases GCN2 and mTORC1 while increasing autocrine and systemic production of fibroblast growth factor 21. This model appears to clarify the role of various risk factors and protective factors in pancreatic cancer induction. A vegan or quasi-vegan low-protein diet may have broader potential for decreasing risk for a range of common “Western” cancers.

PTEN deficiency leads to proteasome addiction: a novel vulnerability in glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months; therefore, more effective treatment options for GBM are required. To identify new drugs targeting GBMs, we performed a high-throughput drug screen using patient-derived neurospheres cultured to preferentially retain their glioblastoma stem cell (GSC) phenotype. High-throughput drug screening was performed on GSCs followed by a dose-response assay of the 5 identified original "hits." A PI3K/mTOR dependency to a proteasome inhibitor (carfilzomib), was confirmed by genetic and pharmacologic experiments. Proteasome Inhibition Response Signatures were derived from proteomic and bioinformatic analysis. Molecular mechanism of action was determined using three-dimensional (3D) GBM-organoids and preclinical orthotopic models. We found that GSCs were highly sensitive to proteasome inhibition due to an underlying dependency on an increased protein synthesis rate, and loss of autophagy, associated with PTEN loss and activation of the PI3K/mTOR pathway. In contrast, combinatory inhibition of autophagy and the proteasome resulted in enhanced cytotoxicity specifically in GSCs that did express PTEN. Finally, proteasome inhibition specifically increased cell death markers in 3D GBM-organoids, suppressed tumor growth, and increased survival of mice orthotopically engrafted with GSCs. As perturbations of the PI3K/mTOR pathway occur in nearly 50% of GBMs, these findings suggest that a significant fraction of these tumors could be vulnerable to proteasome inhibition. Proteasome inhibition is a potential synthetic lethal therapeutic strategy for GBM with proteasome addiction due to a high protein synthesis rate and autophagy deficiency.

Pharmacologic Normalization of Pancreatic Cancer-Associated Fibroblast Secretome Impairs Prometastatic Cross-Talk With Macrophages.

Background & AimsCancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein–coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors.MethodsLarge-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database.ResultsProteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms.ConclusionsWe propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.

Endoplasmic reticulum stress in human chronic wound healing: Rescue by 4-phenylbutyrate.

During wound healing, cells have a high rate of protein synthesis and many proteins need to be folded post‐translationally to function, which occurs in the endoplasmic reticulum (ER). In addition to proliferation, several cellular stress conditions, such as hypoxia, in the wound micro‐environment lead to the accumulation of unfolded or misfolded proteins in the ER, causing ER stress. Eukaryotic cells have a signalling system to manage ER stress called the unfolded protein response (UPR). Mild UPR activation has a beneficial homeostatic effect; however, excessive UPR induces cell death. Herein, we examined venous leg ulcer biopsies versus normal acute incisional wounds in age‐matched elderly subjects and found a large increase in ER stress markers. To study the underlying mechanism, we established several cell cultures from amputated legs from the elderly that showed inherent ER stress. While both keratinocytes and fibroblasts migration was impaired by ER stress, migration of elderly leg skin keratinocytes was markedly improved after treatment with the chemical chaperone and clinically established drug 4‐phenylbutyrate (4‐PBA) and demonstrated a reduction in ER stress markers. In a full‐thickness human skin wound healing model, 4‐PBA improved the reepithelialisation rate, which suggests it as a promising drug repurposing candidate for wound healing.

The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC-MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell-cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Testing the Growth Rate Hypothesis in Two Wetland Macrophytes Under Different Water Level and Sediment Type Conditions.

The growth rate hypothesis (GRH) states that a negative correlation exists between the growth rate and N:P and C:P ratios, because fast-growing organisms need relatively more phosphorus-rich RNA to support their high rates of protein synthesis. However, it is still uncertain whether the GRH is applicable in freshwater wetlands. Several studies have shown that water level and sediment type are key factors influencing plant growth and plant C:N:P characteristics in freshwater wetlands. Thus, this study aimed to elucidate the influence of these factors on plant growth and test the GRH under varying water levels and sediment conditions. We designed a controlled experiment at three water levels and under three sediment types using the two dominant plants (Carex brevicuspis and Polygonum hydropiper) in the East Dongting Lake wetland, and we further investigated the relative growth rate (RGR); concentrations of total carbon (TC), total nitrogen (TN), and total phosphorus (TP); and plant stoichiometry (ratios of C:N, C:P, and N:P) in the aboveground and belowground parts and whole plants in both species. Results demonstrated that the RGR and TC of both species decreased significantly with decreasing sediment nutrient supply and increasing water level. However, TN and TP of both species were markedly higher at high water levels than at low water levels; furthermore, these were significantly higher on clay than on the other two sediment types at each water level. The C:N and C:P ratios of both species decreased with increasing sediment nutrient supply and water level, whereas N:P decreased in both species with increasing sediment nutrient supply. The aboveground part of C. brevicuspis as well as the aboveground part and whole plant of P. hydropiper were negatively correlated with N:P, which is consistent with the GRH. However, the relationship between the belowground RGR and N:P of these species was inconsistent with GRH. Therefore, the water level and sediment type and their interaction significantly influenced plant RGR and C:N:P characteristics. The RGR and plant stoichiometry differed significantly between plant organs, indicating that the GRH needs refinement when applied to wetland macrophytes.

Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1α Axis.

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1α/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1α by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1α (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.