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Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.
© 2019 by The American Society of Hematology.
The emergence of the basement membrane (BM), a specialized form of extracellular matrix, was essential in the unicellular transition to multicellularity. However, the mechanism is unknown. Goodpasture antigen-binding protein (GPBP), a BM protein, was uniquely poised to play diverse roles in this transition owing to its multiple isoforms (GPBP-1, -2, and -3) with varied intracellular and extracellular functions (ceramide trafficker and protein kinase). We sought to determine the evolutionary origin of GPBP isoforms. Our findings reveal the presence of GPBP in unicellular protists, with GPBP-2 as the most ancient isoform. In vertebrates, GPBP-1 assumed extracellular function that is further enhanced by membrane-bound GPBP-3 in mammalians, whereas GPBP-2 retained intracellular function. Moreover, GPBP-2 possesses a dual intracellular/extracellular function in cnidarians, an early nonbilaterian group. We conclude that GPBP functioning both inside and outside the cell was of fundamental importance for the evolutionary transition to animal multicellularity and tissue evolution.
© 2019 Darris et al.
Dehydroalanine (DHA) and dehydrobutyrine (DHB) intermediates, formed through β-elimination, induce protein irreversible glutathionylation and protein-protein crosslinking in human lens fiber cells. In total, irreversible glutathionylation was detected on 52 sites including cysteine, serine and threonine residues in 18 proteins in human lenses. In this study, the levels of GSH modification on three serine residues and four cysteine residues located in seven different lens proteins isolated from different regions and different aged lenses were quantified. The relative levels of modification (modified/nonmodified) were site-specific and age-related, ranging from less than 0.05% to about 500%. The levels of modification on all of the sites quantified in the lens cortex increased with age and GSH modification also increased from cortex to outer nucleus region suggesting an age-related increase of modification. The levels of modification on sites located in stable regions of the proteins such as Cys117 of βA3, Cys80 of βB1 and Cys27 of γS, continued increasing in inner nucleus, but modification on sites located in regions undergoing degradation with age decreased in the inner nucleus suggesting GSH modified proteins were more susceptible to further modification. Irreversible GSH modification in cataract lenses was typically higher than in age-matched normal lenses, but the difference did not reach statistical significance for a majority of sites, with the exception Cys117 of βA3 crystallin in WSF. Except for S59 of αA and αB crystallins, GSH modification did not induce protein insolubility suggesting a possible role for this modification in protection from protein-protein crosslinking.
Copyright © 2018 Elsevier Ltd. All rights reserved.
Herein, excipients are investigated to ameliorate the deleterious effects of lyophilization on peptide-polymer nano-polyplex (NP) morphology, cellular uptake, and bioactivity. The NPs are a previously-described platform technology for intracellular peptide delivery and are formulated from a cationic therapeutic peptide and the anionic, pH-responsive, endosomolytic polymer poly(propylacrylic acid) (PPAA). These NPs are effective when formulated and immediately used for delivery into cells and tissue, but they are not amenable to reconstitution following storage as a lyophilized powder due to aggregation. To develop a lyophilized NP format that facilitates longer-term storage and ease of use, MAPKAP kinase 2 inhibitory peptide-based NPs (MK2i-NPs) were prepared in the presence of a range of concentrations of the excipients sucrose, trehalose, and lactosucrose prior to lyophilization and storage. All excipients improved particle morphology post-lyophilization and significantly improved MK2i-NP uptake in human coronary artery smooth muscle cells relative to lyophilized NPs without excipient. In particular, MK2i-NPs lyophilized with 300 mM lactosucrose as an excipient demonstrated a 5.23 fold increase in cellular uptake (p < 0.001), a 2.52 fold increase in endosomal disruption (p < 0.05), and a 2.39 fold increase in ex vivo bioactivity (p < 0.01) compared to MK2i-NPs lyophilized without excipients. In sum, these data suggest that addition of excipients, particularly lactosucrose, maintains and even improves the uptake and therapeutic efficacy of peptide-polymer NPs post-lyophilization relative to freshly-made formulations. Thus, the use of excipients as lyoprotectants is a promising approach for the long-term storage of biotherapeutic NPs and poises this NP platform for clinical translation.
Copyright © 2018 Elsevier B.V. All rights reserved.
Monophosphoryl lipid A (MPLA) is a clinically used TLR4 agonist that has been found to drive nonspecific resistance to infection for up to 2 wk. However, the molecular mechanisms conferring protection are not well understood. In this study, we found that MPLA prompts resistance to infection, in part, by inducing a sustained and dynamic metabolic program in macrophages that supports improved pathogen clearance. Mice treated with MPLA had enhanced resistance to infection with and that was associated with augmented microbial clearance and organ protection. Tissue macrophages, which exhibited augmented phagocytosis and respiratory burst after MPLA treatment, were required for the beneficial effects of MPLA. Further analysis of the macrophage phenotype revealed that early TLR4-driven aerobic glycolysis was later coupled with mitochondrial biogenesis, enhanced malate shuttling, and increased mitochondrial ATP production. This metabolic program was initiated by overlapping and redundant contributions of MyD88- and TRIF-dependent signaling pathways as well as downstream mTOR activation. Blockade of mTOR signaling inhibited the development of the metabolic and functional macrophage phenotype and ablated MPLA-induced resistance to infection in vivo. Our findings reveal that MPLA drives macrophage metabolic reprogramming that evolves over a period of days to support a macrophage phenotype highly effective at mediating microbe clearance and that this results in nonspecific resistance to infection.
Copyright © 2018 by The American Association of Immunologists, Inc.
The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.
Copyright © 2017 Elsevier Inc. All rights reserved.
SPAK (STE20/SPS1-related proline/alanine-rich kinase) regulates Na and Cl reabsorption in the distal convoluted tubule, and possibly in the thick ascending limb of Henle. This kinase phosphorylates and activates the apical Na-Cl cotransporter in the DCT. Western blot analysis reveals that SPAK in kidney exists as a full-length protein as well as shorter fragments that might affect NKCC2 function in the TAL. Recently, we showed that kidney lysates exerts proteolytic activity towards SPAK, resulting in the formation of multiple SPAK fragments with possible inhibitory effects on the kinase. The proteolytic activity is mediated by a Zn metalloprotease inhibited by 1,10-phenanthroline, DTT, and EDTA. Size exclusion chromatography demonstrated that the protease was a high-molecular-weight protein. Protein identification by mass-spectrometry analysis after ion exchange and size exclusion chromatography identified multiple proteases as possible candidates and aspartyl aminopeptidase, DNPEP, shared all the properties of the kidney lysate activity. Furthermore, recombinant GST-DNPEP produced similar proteolytic pattern. No mouse knockout model was, however, available to be used as negative control. In this study, we used a DNPEP-mutant mouse generated by EUCOMM as well as a novel CRISPR/cas9 mouse knockout to assess the activity of their kidney lysates towards SPAK. Two mouse models had to be used because different anti-DNPEP antibodies provided conflicting data on whether the EUCOMM mouse resulted in a true knockout. We show that in the absence of DNPEP, the kidney lysates retain their ability to cleave SPAK, indicating that DNPEP might have been misidentified as the protease behind the kidney lysate activity, or that the aspartyl aminopeptidase might not be the only protease cleaving SPAK in kidney.
© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.
Cerebellar growth and foliation require the Hedgehog-driven proliferation of granule cell precursors (GCPs) in the external granule layer (EGL). However, that increased or extended GCP proliferation generally does not elicit ectopic folds suggests that additional determinants control cortical expansion and foliation during cerebellar development. Here, we find that genetic loss of the serine-threonine kinase Liver Kinase B1 (Lkb1) in GCPs increased cerebellar cortical size and foliation independent of changes in proliferation or Hedgehog signaling. This finding is unexpected given that Lkb1 has previously shown to be critical for Hedgehog pathway activation in cultured cells. Consistent with unchanged proliferation rate of GCPs, the cortical expansion of Lkb1 mutants is accompanied by thinning of the EGL. The plane of cell division, which has been implicated in diverse processes from epithelial surface expansions to gyrification of the human cortex, remains unchanged in the mutants when compared to wild-type controls. However, we find that Lkb1 mutants display delayed radial migration of post-mitotic GCPs that coincides with increased cortical size, suggesting that aberrant cell migration may contribute to the cortical expansion and increase foliation. Taken together, our results reveal an important role for Lkb1 in regulating cerebellar cortical size and foliation in a Hedgehog-independent manner.
Copyright © 2017 Elsevier Inc. All rights reserved.
Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.
© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com.