The Endoplasmic Reticulum: A Hub of Protein Quality Control in Health and Disease

Publication date: Available online 29 March 2017
Source:Free Radical Biology and Medicine
Author(s): Lisa Vincenz-Donnelly, Mark S. Hipp
One third of the eukaryotic proteome is synthesized at the endoplasmic reticulum (ER), whose unique properties provide a folding environment substantially different from the cytosol. A healthy, balanced proteome in the ER is maintained by a network of factors referred to as the ER quality control (ERQC) machinery. This network consists of various protein folding chaperones and modifying enzymes, and is regulated by stress response pathways that prevent the build-up as well as the secretion of potentially toxic and aggregation-prone misfolded protein species. Here, we describe the components of the ERQC machinery, investigate their response to different forms of stress, and discuss the consequences of ERQC break-down.

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Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production

Publication date: Available online 29 March 2017
Source:Free Radical Biology and Medicine
Author(s): Natália P Nogueira, Francis MS Saraiva, Matheus P Oliveira, Ana Paula M Mendonça, Job D.F. Inacio, Elmo E Almeida-Amaral, Rubem F Menna-Barreto, Gustavo AT Laranja, Eduardo J Lopes Torres, Marcus F Oliveira, Marcia C Paes
Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)−2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation.

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Methylglyoxal-induced AMPK activation leads to autophagic degradation of thioredoxin 1 and glyoxalase 2 in HT22 nerve cells

Publication date: Available online 29 March 2017
Source:Free Radical Biology and Medicine
Author(s): Alcir Luiz Dafre, Ariana Ern Schmitz, Pamela Maher
Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. We previously showed that MGO treatment targets the thioredoxin and the glyoxalase systems, leading to a decrease in Trx1 and Glo2 proteins in immortalized mouse hippocampal HT22 nerve cells. Here, we propose that autophagy is the underlying mechanism leading to Glo2 and Trx1 loss induced by MGO. The autophagic markers p62, and the lipidated and active form of LC3, were increased by MGO (0.5mM). Autophagy inhibition with bafilomycin or chloroquine prevented the decrease in Trx1 and Glo2 at 6 and 18h after MGO treatment. Proteasome inhibition by MG132 exacerbated the effect of MGO on Trx1 and Glo2 degradation (18h), further suggesting a role for autophagy. ATG5 small interfering RNA protected Trx1 and Glo2 from MGO-induced degradation, confirming Trx1 and Glo2 loss is mediated by autophagy. In the search for the signals that control autophagy, we found that AMPK activation, a known autophagy inducer, was markedly increased by MGO treatment. AMPK activation was confirmed by increased acetyl coenzyme A carboxylase phosphorylation, a direct AMPK substrate and by decreased mTOR phosphorylation, an indirect marker of AMPK activation. To confirm that MGO-mediated Trx1 and Glo2 degradation was AMPK-dependent, AMPK-deficient mouse embryonic fibroblasts (MEFs) were treated with MGO. Wildtype MEFs presented the expected decrease in Trx1 and Glo2, while MGO was ineffective in decreasing these proteins in AMPK-deficient cells. Overall, the data indicate that MGO activates autophagy in an AMPK-dependent manner, and that autophagy was responsible for Trx1 and Glo2 degradation, confirming that Trx1 and Glo2 are molecular targets of MGO.

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Zinc Regulates Nox1 Expression Through a NF-κB and Mitochondrial ROS Dependent Mechanism to Induce Senescence of Vascular Smooth Muscle Cells

Publication date: Available online 29 March 2017
Source:Free Radical Biology and Medicine
Author(s): G. Salazar, J. Huang, R.G. Feresin, Y. Zhao, K.K. Griendling
AimsThe role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc-dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined.ResultsHere, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II- and zinc-induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF-κB, a known Nox1 transcriptional regulator and by N-acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF-κB and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF-κB activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc-induced senescence was reduced by inhibition of NF-κB or reduction of mitochondrial ROS with MitoTEMPO. NF-κB activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF-κB.Innovation and ConclusionOur data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF-κB activation which in turn upregulates Nox1 expression inducing senescence of VSMCs.

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Controversies in clinicopathological characteristics and treatment strategies of male breast cancer: A review of the literature

Publication date: Available online 29 March 2017
Source:Critical Reviews in Oncology/Hematology
Author(s): Agnese Losurdo, Selene Rota, Giuseppe Gullo, Giovanna Masci, Rosalba Torrisi, Giulia Bottai, Monica Zuradelli, Wolfgang Gatzemeier, Armando Santoro
Male breast cancer (MaBC) is a rare disease, accounting for less than 1% of malignancies in men. For this reason, literature data on its clinicopathological characteristics are very heterogeneous and treatment strategies have mostly been extrapolated from the female counterpart. However, immunohistochemical peculiarities of MaBC have recently emerged, defining it as a distinct entity from female breast cancer (FBC), thus requiring a tailored clinical approach. MaBC appears to be more often hormone receptor positive than FBC, while data on HER2 status still remain inconclusive, indicating a possible higher incidence of HER2 alterations.Treatment strategies for MaBC have evolved and less invasive local treatments such as lumpectomy and sentinel lymph node biopsy have become part of everyday clinical practice, while there are still controversies on the indication of radiotherapy, especially after mastectomy. Similarly, differences between male and female hormonal status have raised some concerns in the use of aromatase inhibitors in male patients and the choice of best endocrine therapy is still controversial.



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Treatment Options for Metastatic Colorectal Cancer in Patients with Liver Dysfunction due to Malignancy

Publication date: Available online 29 March 2017
Source:Critical Reviews in Oncology/Hematology
Author(s): L. Faugeras, A. Dili, A. Druez, B. Krug, Decoster, L. D'Hondt
BackgroundThe survival of colorectal cancer patients is frequently determined by the extent of metastatic invasion to the liver; in cases of major involvement, therapeutic strategies are limited because the liver is necessary for drug metabolism.Material and methodsWe have reviewed articles about the pharmacokinetic profiles of each drug used in colorectal cancer patients with hepatic dysfunction to determine which of these treatments are most feasible.ResultsSome drugs appear to be feasible options for patients with hepatic insufficiency. Agents such as 5-fluorouracil and oxaliplatin, as well as monoclonal antibodies such as bevacizumab, cetuximab, and panitumumab, can potentially be used in these cases. On the other hand, irinotecan and regorafenib cannot be recommended because of the risk of increased toxicity.ConclusionTreatment of patients with colorectal cancer and liver dysfunction represents a major challenge because the prognosis is usually very poor and alteration of liver function is normally an exclusion criterion in clinical trials. In this review, we present evidence regarding the use of each drug in patients with colorectal cancer and hepatic impairment.



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Protein separation through preliminary experiments concerning pH and salt concentration by tube radial distribution chromatography based on phase separation multiphase flow using a polytetrafluoroethylene capillary tube

Publication date: 1 July 2017
Source:Talanta, Volume 169
Author(s): Hyo Kan, Kazuhiko Tsukagoshi
Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water–acetonitrile–ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer–acetonitrile–ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution.



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A quantitative method for detecting DNA methylation over targeted genomic regions using isotope dilution liquid chromatography tandem mass spectrometry

Publication date: 1 July 2017
Source:Talanta, Volume 169
Author(s): Xiaoxia Ye, Lijian Zhang, Bin Chen, Jian Li, Qingjin Yang, Qionglin Huang, Junjie Zhang, Yihua Gao, Zhaojin Li, Chun Cai
Aberrant DNA methylation is associated with various diseases. Quantitative analysis of regional DNA methylation levels of some specific genes would aid in diseases diagnosis and risk stratification. In this study, we developed a robust method for detecting DNA methylation level over targeted genomic regions using nucleobases quantification in bisulfite amplicons by isotope dilution liquid chromatography tandem mass spectrometry coupled with a simple equation. This method had wide detection range (from 0% to 100% methylation) and high accuracy while more time-saving compared to clonal bisulfite sequencing method. The application for clinical tissue samples showed good applicability and cost effectiveness. This analytical method is suitable for quantifying average DNA methylation level over targeted genomic regions and expected to be a useful tool for detecting DNA methylation biomarkers.

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Liver-Specific Activation of AMPK Prevents Steatosis on a High-Fructose Diet

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Angela Woods, Jennet R. Williams, Phillip J. Muckett, Faith V. Mayer, Maria Liljevald, Mohammad Bohlooly-Y, David Carling
AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. We identified a mutation in the γ1 subunit (γ1^D316A) that leads to activation of AMPK. We generated mice with this mutation to study the effect of chronic liver-specific activation of AMPK in vivo. Primary hepatocytes isolated from these mice have reduced gluconeogenesis and fatty acid synthesis, but there is no effect on fatty acid oxidation compared to cells from wild-type mice. Liver-specific activation of AMPK decreases lipogenesis in vivo and completely protects against hepatic steatosis when mice are fed a high-fructose diet. Our findings demonstrate that liver-specific activation of AMPK is sufficient to protect against hepatic triglyceride accumulation, a hallmark of non-alcoholic fatty liver disease (NAFLD). These results emphasize the clinical relevance of activating AMPK in the liver to combat NAFLD and potentially other associated complications (e.g., cirrhosis and hepatocellular carcinoma).

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Using a gain-of-function mouse model, Woods et al. show that hepatic activation of AMPK protects against triglyceride accumulation in the liver. AMPK inhibits de novo lipogenesis but has no effect on hepatic fatty acid oxidation. These findings highlight AMPK as an attractive therapeutic target for protection against fatty liver disease.


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Neuroprotective Functions for the Histone Deacetylase SIRT6

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Shai Kaluski, Miguel Portillo, Antoine Besnard, Daniel Stein, Monica Einav, Lei Zhong, Uwe Ueberham, Thomas Arendt, Raul Mostoslavsky, Amar Sahay, Debra Toiber
The histone deacetylase SIRT6 promotes DNA repair, but its activity declines with age with a concomitant accumulation of DNA damage. Furthermore, SIRT6 knockout mice exhibit an accelerated aging phenotype and die prematurely. Here, we report that brain-specific SIRT6-deficient mice survive but present behavioral defects with major learning impairments by 4 months of age. Moreover, the brains of these mice show increased signs of DNA damage, cell death, and hyperphosphorylated Tau—a critical mark in several neurodegenerative diseases. Mechanistically, SIRT6 regulates Tau protein stability and phosphorylation through increased activation of the kinase GSK3α/β. Finally, SIRT6 mRNA and protein levels are reduced in patients with Alzheimer's disease. Taken together, our results suggest that SIRT6 is critical to maintain genomic stability in the brain and that its loss leads to toxic Tau stability and phosphorylation. Therefore, SIRT6 and its downstream signaling could be targeted in Alzheimer's disease and age-related neurodegeneration.

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Kaluski et al. show that lack of SIRT6 in the brain provokes neurodegeneration by increasing DNA damage, apoptosis, and toxic Tau phosphorylation. DNA damage or lack of SIRT6 activates GSK3, resulting in Tau phosphorylation and stability. GSK3 inhibition rescues this phenotype. Importantly, AD patients show reduced SIRT6 in the brain.


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Altered Synapse Stability in the Early Stages of Tauopathy

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Johanna S. Jackson, Jonathan Witton, James D. Johnson, Zeshan Ahmed, Mark Ward, Andrew D. Randall, Michael L. Hutton, John T. Isaac, Michael J. O'Neill, Michael C. Ashby
Synapse loss is a key feature of dementia, but it is unclear whether synaptic dysfunction precedes degenerative phases of the disease. Here, we show that even before any decrease in synapse density, there is abnormal turnover of cortical axonal boutons and dendritic spines in a mouse model of tauopathy-associated dementia. Strikingly, tauopathy drives a mismatch in synapse turnover; postsynaptic spines turn over more rapidly, whereas presynaptic boutons are stabilized. This imbalance between pre- and post-synaptic stability coincides with reduced synaptically driven neuronal activity in pre-degenerative stages of the disease.

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Using in vivo two-photon imaging in the rTg4510 tauopathy mouse model, Jackson et al. find that synapse stability is altered during the pre-degenerative stages of tauopathy. Mismatched abnormalities in pre- and post-synaptic turnover coincide with disrupted neuronal activity.


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Identification of a Tissue-Restricted Isoform of SIRT1 Defines a Regulatory Domain that Encodes Specificity

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Shaunak Deota, Tandrika Chattopadhyay, Deepti Ramachandran, Eric Armstrong, Beatriz Camacho, Babukrishna Maniyadath, Amit Fulzele, Anne Gonzalez-de-Peredo, John M. Denu, Ullas Kolthur-Seetharam
The conserved NAD⁺-dependent deacylase SIRT1 plays pivotal, sometimes contrasting, roles in diverse physiological and pathophysiological conditions. In this study, we uncover a tissue-restricted isoform of SIRT1 (SIRT1-ΔE2) that lacks exon 2 (E2). Candidate-based screening of SIRT1 substrates demonstrated that the domain encoded by this exon plays a key role in specifying SIRT1 protein-protein interactions. The E2 domain of SIRT1 was both necessary and sufficient for PGC1α binding, enhanced interaction with p53, and thus downstream functions. Since SIRT1-FL and SIRT1-ΔE2 were found to have similar intrinsic catalytic activities, we propose that the E2 domain tethers specific substrate proteins. Given the absence of SIRT1-ΔE2 in liver, our findings provide insight into the role of the E2 domain in specifying "metabolic functions" of SIRT1-FL. Identification of SIRT1-ΔE2 and the conserved specificity domain will enhance our understanding of SIRT1 and guide the development of therapeutic interventions.

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Deota et al. identify SIRT1-ΔE2, a tissue-restricted SIRT1 isoform, and a conserved domain within SIRT1 that results in specific interactions. This domain is linked to metabolic homeostasis and DNA damage response and may point to strategies for precise modulation of SIRT1.


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Pneumocystis-Driven Inducible Bronchus-Associated Lymphoid Tissue Formation Requires Th2 and Th17 Immunity

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Taylor Eddens, Waleed Elsegeiny, Maria de la Luz Garcia-Hernadez, Patricia Castillo, Giraldina Trevejo-Nunez, Katelin Serody, Brian T. Campfield, Shabaana A. Khader, Kong Chen, Javier Rangel-Moreno, Jay K. Kolls
Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid structure composed of highly organized T cell and B cell zones that forms in the lung in response to infectious or inflammatory stimuli. Here, we develop a model for fungal-mediated iBALT formation, using infection with Pneumocystis that induces development of pulmonary lymphoid follicles. Pneumocystis-dependent iBALT structure formation and organization required CXCL13 signaling. Cxcl13 expression was regulated by interleukin (IL)-17 family members, as Il17ra^−/−, Il17rb^−/−, and Il17rc^−/− mice failed to develop iBALT. Interestingly, Il17rb^−/− mice have intact Th17 responses, but failed to generate an anti-Pneumocystis Th2 response. Given a role for Th2 and Th17 immunity in iBALT formation, we demonstrated that primary pulmonary fibroblasts synergistically upregulated Cxcl13 transcription following dual stimulation with IL-13 and IL-17A in a STAT3/GATA3-dependent manner. Together, these findings uncover a role for Th2/Th17 cells in regulating Cxcl13 expression and provide an experimental model for fungal-driven iBALT formation.

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Eddens et al. develop a model for fungal-inducible bronchus-associated lymphoid tissue (iBALT) formation driven by infection or exposure to Pneumocystis. Pneumocystis induces Th2 and Th17 immunity, both of which are required for iBALT formation.


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Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Marija Maric, Progya Mukherjee, Michael H. Tatham, Ronald Hay, Karim Labib
Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 "segregase". Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.

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Disassembly of the CMG helicase is the key regulated step during DNA replication termination in eukaryotes, driven by CMG ubiquitylation and the Cdc48 segregase. Maric et al. find that the Ufd1-Npl4 heterodimer is essential for CMG disassembly in budding yeast and is required to recruit Cdc48 to the ubiquitylated helicase.


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Th1 Differentiation Drives the Accumulation of Intravascular, Non-protective CD4 T Cells during Tuberculosis

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Michelle A. Sallin, Shunsuke Sakai, Keith D. Kauffman, Howard A. Young, Jinfang Zhu, Daniel L. Barber
Recent data indicate that the differentiation state of Th1 cells determines their protective capacity against tuberculosis. Therefore, we examined the role of Th1-polarizing factors in the generation of protective and non-protective subsets of Mtb-specific Th1 cells. We find that IL-12/23p40 promotes Th1 cell expansion and maturation beyond the CD73⁺CXCR3⁺T-bet^dim stage, and T-bet prevents deviation of Th1 cells into Th17 cells. Nevertheless, IL- 12/23p40 and T-bet are also essential for the production of a prominent subset of intravascular CX3CR1⁺KLRG1⁺ Th1 cells that persists poorly and can neither migrate into the lung parenchyma nor control Mtb growth. Furthermore, T-bet suppresses development of CD69⁺CD103⁺ tissue resident phenotype effectors in lung. In contrast, Th1-cell-derived IFN-γ inhibits the accumulation of intravascular CX3CR1⁺KLRG1⁺ Th1 cells. Thus, although IL-12 and T-bet are essential host survival factors, they simultaneously oppose lung CD4 T cell responses at several levels, demonstrating the dual nature of Th1 polarization in tuberculosis.

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Sallin et al. show that IL-12/23p40 and T-bet have a detrimental impact on the quality of some Th1 cells in TB. Although required for IFN-γ production and host survival, these factors are dispensable for migration of T cells into the lung and drive the differentiation of non-protective intravascular CX3CR1⁺KLRG1⁺ Th1 cells.


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Cell Traversal Activity Is Important for Plasmodium falciparum Liver Infection in Humanized Mice

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Annie S.P. Yang, Matthew T. O'Neill, Charlie Jennison, Sash Lopaticki, Cody C. Allison, Jennifer S. Armistead, Sara M. Erickson, Kelly L. Rogers, Andrew M. Ellisdon, James C. Whisstock, Rebecca E. Tweedell, Rhoel R. Dinglasan, Donna N. Douglas, Norman M. Kneteman, Justin A. Boddey
Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen.

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Malaria sporozoites deposited by a mosquito into the skin must navigate to the liver and infect hepatocytes. Yang et al. reveal the importance of sporozoite transmigration through host cells to establish liver infection in humanized mice by the most virulent human malaria parasite Plasmodium falciparum.


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Identification of Interleukin-1 by Functional Screening as a Key Mediator of Cellular Expansion and Disease Progression in Acute Myeloid Leukemia

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Alyssa Carey, David K. Edwards, Christopher A. Eide, Laura Newell, Elie Traer, Bruno C. Medeiros, Daniel A. Pollyea, Michael W. Deininger, Robert H. Collins, Jeffrey W. Tyner, Brian J. Druker, Grover C. Bagby, Shannon K. McWeeney, Anupriya Agarwal
Secreted proteins in the bone marrow microenvironment play critical roles in acute myeloid leukemia (AML). Through an ex vivo functional screen of 94 cytokines, we identified that the pro-inflammatory cytokine interleukin-1 (IL-1) elicited profound expansion of myeloid progenitors in ∼67% of AML patients while suppressing the growth of normal progenitors. Levels of IL-1β and IL-1 receptors were increased in AML patients, and silencing of the IL-1 receptor led to significant suppression of clonogenicity and in vivo disease progression. IL-1 promoted AML cell growth by enhancing p38MAPK phosphorylation and promoting secretion of various other growth factors and inflammatory cytokines. Treatment with p38MAPK inhibitors reversed these effects and recovered normal CD34⁺ cells from IL-1-mediated growth suppression. These results highlight the importance of ex vivo functional screening to identify common and actionable extrinsic pathways in genetically heterogeneous malignancies and provide impetus for clinical development of IL-1/IL1R1/p38MAPK pathway-targeted therapies in AML.

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Carey et al. show that most AML patients are dependent on IL-1 and suggest that an IL-1-rich environment promotes the expansion of AML progenitors while suppressing normal progenitors by differently influencing cell proliferation, survival, and differentiation. AML patients with aberrant IL-1 signaling may benefit from therapeutically targeting this pathway to enhance normal hematopoiesis while inhibiting AML.


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SMARCAD1 Contributes to the Regulation of Naive Pluripotency by Interacting with Histone Citrullination

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Shu Xiao, Jia Lu, Bharat Sridhar, Xiaoyi Cao, Pengfei Yu, Tianyi Zhao, Chieh-Chun Chen, Darina McDee, Laura Sloofman, Yang Wang, Marcelo Rivas-Astroza, Bhanu Prakash V.L. Telugu, Dana Levasseur, Kang Zhang, Han Liang, Jing Crystal Zhao, Tetsuya S. Tanaka, Gary Stormo, Sheng Zhong
Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation.

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Xiao et al. identify SMARCAD1 as a candidate reader protein of the H3R26Cit histone modification. Knockdown of Smarcad1 causes mouse ESCs to exit the naive pluripotent state and leads to increased H3K9me3 at SMARCAD1-binding regions. This suggests a model whereby SMARCAD1 guards naive pluripotency by interacting with H3R26Cit and suppressing H3K9me3.


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Coordinated Pulses of mRNA and of Protein Translation or Degradation Produce EGF-Induced Protein Bursts

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Roni Golan-Lavi, Chiara Giacomelli, Garold Fuks, Amit Zeisel, Johanna Sonntag, Sanchari Sinha, Wolfgang Köstler, Stefan Wiemann, Ulrike Korf, Yosef Yarden, Eytan Domany
Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles.

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Golan-Lavi et al. show that the cellular response to an external stimulus generates rapid transient bursts of abundance of some proteins. Integrating time-dependent transcript and protein data into a mathematical model, they infer complex temporal protein degradation profiles, acting in concert with an mRNA pulse to produce the protein burst.


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Enhanced Degradation of Misfolded Proteins Promotes Tumorigenesis

Publication date: 28 March 2017
Source:Cell Reports, Volume 18, Issue 13
Author(s): Liang Chen, Michael D. Brewer, Lili Guo, Ruoxing Wang, Peng Jiang, Xiaolu Yang
An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.

Graphical abstract

Teaser

A diminished cellular capacity to degrade misfolded proteins contributes to aging and neurodegeneration. Chen et al. report that a heightened degradation capacity promotes tumorigenesis. Upregulation of the proteasome and TRIM proteins in tumor cells supports enhanced degradation of misfolded proteins, which bolsters antioxidant defense during oncogenic growth.


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