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Protein lifespan is regulated by co-translational modification by several enzymes, including methionine aminopeptidases and N-alpha-aminoterminal acetyltransferases. The NatB enzymatic complex is an N-terminal acetyltransferase constituted by two subunits, NAA20 and NAA25, whose interaction is necessary to avoid NAA20 catalytic subunit degradation. We found that deletion of the first five amino acids of hNAA20 or fusion of a peptide to its amino terminal end abolishes its interaction with hNAA25. Substitution of the second residue of hNAA20 with amino acids with small, uncharged side-chains allows NatB enzymatic complex formation. However, replacement by residues with large or charged side-chains interferes with its hNAA25 interaction, limiting functional NatB complex formation. Comparison of NAA20 eukaryotic sequences showed that the residue following the initial methionine, an amino acid with a small uncharged side-chain, has been evolutionarily conserved. We have confirmed the relevance of second amino acid characteristics of NAA20 in NatB enzymatic complex formation in Drosophila melanogaster. Moreover, we have evidenced the significance of NAA20 second residue in Saccharomyces cerevisiae using different NAA20 versions to reconstitute NatB formation in a yNAA20-KO yeast strain. The requirement in humans and in fruit flies of an amino acid with a small uncharged side-chain following the initial methionine of NAA20 suggests that methionine aminopeptidase action may be necessary for the NAA20 and NAA25 interaction. We showed that inhibition of MetAP2 expression blocked hNatB enzymatic complex formation by retaining the initial methionine of NAA20. Therefore, NatB-mediated protein N-terminal acetylation is dependent on methionine aminopeptidase, providing a regulatory mechanism for protein N-terminal maturation.

Contains fulltext : 225281.pdf (Publisher’s version ) (Closed access) Contains fulltext : 225281pos.pdf (Author’s version postprint ) (Open Access) BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. METHODS: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. RESULTS: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. CONCLUSIONS: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

Criticism of the Centers for Disease Control and Prevention (CDC) and its efforts to put forth recommendations and data based on core public health practice and principles is contrary to the realities of the work that the CDC has done on preparedness in past decades In 2001, the CDC funded a project intended to facilitate the development of an integrated national system of Academic Centers for Public Health Preparedness (CPHPs) to train frontline public health professionals who respond to bioterrorist incidents and other emerging infectious diseases Nineteen CPHPs were funded until the program was discontinued in August 2010 and replaced by 14 Preparedness and Emergency Response Learning Centers, whose funding ended in 2018 2 CPHPs worked to identify core principles for public health preparedness and response, strategies for local and state data collection, and curricula and best practices for educating the public health and first responder workforce

CONTEXT In March, 2020, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), was spreading in the Bay Area, especially in Santa Clara County, causing increases in cases, hospitalizations, and deaths. PROGRAM The Association of Bay Area Health Officials (ABAHO) represents 13 Bay Area health jurisdictions. IMPLEMENTATION On March 15, 2020, the local health officers of 7 ABAHO members (counties of Alameda, Contra Costa, Marin, San Francisco, San Mateo, and Santa Clara and the city of Berkeley) decided to issue legal orders on March 16 for 6.7 million residents to shelter in place to prevent the spread of SARS-CoV-2, the causal agent of COVID-19. The Bay Area was the first region in the United States to shelter in place, and within days, other regions in the United States followed. EVALUATION Subsequent comparative analyses have confirmed that acting early in issuing shelter-in-place orders prevented a large number of cases, hospitalizations, and deaths in the Bay Area throughout the United States. The quality of a decision-in this case, for crisis decision making-cannot be judged by the outcome. A good decision can have a bad outcome, and a bad decision can have a good outcome. The quality of a decision depends only on the quality of the decision-making process at the time the decision was made. DISCUSSION In this Field Report, we review how we made this collective decision. With the benefit of hindsight and reflection, we recount our story through the lens of public health legal authority, meta-leadership, and decision intelligence. Our purpose is to improve the crisis decision-making skills of public health officials by improving how we make high-stakes decisions each day in our continuing fight to contain the SARS-CoV-2 pandemic, to save lives, and to eliminate COVID-19 racial/ethnic inequities.

Tight control of transgene expression is key to ensure the efficacy of a wide range of gene therapy interventions, in which the magnitude and duration of gene expression have to be adjusted to therapeutic needs, thereby limiting secondary effects. The development of upgraded strategies to link transgene expression to pathological stress episodes is an unmet need in gene therapy. Here, we propose an expression strategy that associates transgene expression to an intracellular stress coping mechanism, the unfolded protein response. Specifically, we harnessed the

Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Since its discovery more than 25 years ago, great progress has been made in our understanding of the unfolded protein response (UPR), a homeostatic mechanism that adjusts endoplasmic reticulum (ER) function to satisfy the physiological demands of the cell. However, if ER homeostasis is unattainable, the UPR switches to drive cell death to remove defective cells in an effort to protect the health of the organism. This functional dichotomy places the UPR at the crossroads of the adaptation versus apoptosis decision. Here, we focus on new developments in UPR signaling mechanisms, in the interconnectivity among the signaling pathways that make up the UPR in higher eukaryotes, and in the coordination between the UPR and other fundamental cellular processes.

Background & Aims HDV infection induces the most severe form of human viral hepatitis. However, the specific reasons for the severity of the disease remain unknown. Recently, we developed an HDV replication mouse model in which, for the first time, liver damage was detected. Methods HDV and HBV replication-competent genomes and HDV antigens were delivered to mouse hepatocytes using adeno-associated vectors (AAVs). Aminotransferase elevation, liver histopathology, and hepatocyte death were evaluated and the immune infiltrate was characterized. Liver transcriptomic analysis was performed. Mice deficient for different cellular and molecular components of the immune system, as well as depletion and inhibition studies, were employed to elucidate the causes of HDV-mediated liver damage. Results AAV-mediated HBV/HDV coinfection caused hepatocyte necrosis and apoptosis. Activated T lymphocytes, natural killer cells, and proinflammatory macrophages accounted for the majority of the inflammatory infiltrate. However, depletion studies and the use of different knockout mice indicated that neither T cells, natural killer cells nor macrophages were necessary for HDV-induced liver damage. Transcriptomic analysis revealed a strong activation of type I and II interferon (IFN) and tumor necrosis factor (TNF)-α pathways in HBV/HDV-coinfected mice. While the absence of IFN signaling had no effect, the use of a TNF-α antagonist resulted in a significant reduction of HDV-associated liver injury. Furthermore, hepatic expression of HDAg resulted in the induction of severe liver damage, which was T cell- and TNF-α-independent. Conclusions Both host (TNF-α) and viral (HDV antigens) factors play a relevant role in HDV-induced liver damage. Importantly, pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage. Lay summary Chronic hepatitis delta constitutes the most severe form of viral hepatitis. There is limited data on the mechanism involved in hepatitis delta virus (HDV)-induced liver pathology. Our data indicate that a cytokine (TNF-α) and HDV antigens play a relevant role in HDV-induced liver damage., Graphical abstract, Highlights • There is limited data on the mechanisms involved in HDV-induced liver pathology. • Our data indicate that both TNF-α and HDV antigens play a relevant role in HDV-induced liver damage. • Pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage.

Background Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O(6)-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide. Methods MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided. Results Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models. Conclusions These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma.

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH., Alcoholic hepatitis, a common cause of liver failure, lacks effective treatment. Here, the authors show altered hepatic HNF4a isoform expression and hypermethylation of its target genes in patients. HNF4a dysregulation is improved in vitro by TGFb or PPARg modulation suggesting potential therapeutic avenues.

Early life events are associated with the susceptibility to chronic diseases in adult life. Perturbations of endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which contributes to the development of metabolic alterations. Our aim was to evaluate liver UPR in an animal model of intrauterine growth restriction (IUGR). A significantly increased expression of X-box binding protein-1 spliced (XBP1s) mRNA (p

Author(s): Grumbach, Kevin; Vargas, Roberto A; Fleisher, Paula; Aragon, Tomas J; Chung, Lisa; Chawla, Colleen; Yant, Abbie; Garcia, Estela R; Santiago, Amor; Lang, Perry L; Jones, Paula; Liu, Wylie; Schmidt, Laura A | Abstract: BackgroundThe San Francisco Health Improvement Partnership (SFHIP) promotes health equity by using a novel collective impact model that blends community engagement with evidence-to-policy translational science. The model involves diverse stakeholders, including ethnic-based community health equity coalitions, the local public health department, hospitals and health systems, a health sciences university, a school district, the faith community, and others sectors.Community contextWe report on 3 SFHIP prevention initiatives: reducing consumption of sugar sweetened beverages (SSBs), regulating retail alcohol sales, and eliminating disparities in children's oral health.MethodsSFHIP is governed by a steering committee. Partnership working groups for each initiative collaborate to 1) develop and implement action plans emphasizing feasible, scalable, translational-science-informed interventions and 2) consider sustainability early in the planning process by including policy and structural interventions.OutcomeThrough SFHIP's efforts, San Francisco enacted ordinances regulating sale and advertising of SSBs and a ballot measure establishing a soda tax. Most San Francisco hospitals implemented or committed to implementing healthy-beverage policies that prohibited serving or selling SSBs. SFHIP helped prevent Starbucks and Taco Bell from receiving alcohol licenses in San Francisco and helped prevent state authorization of sale of powdered alcohol. SFHIP increased the number of primary care clinics providing fluoride varnish at routine well-child visits from 3 to 14 and acquired a state waiver to allow dental clinics to be paid for dental services delivered in schools.InterpretationThe SFHIP model of collective impact emphasizing community engagement and policy change accomplished many of its intermediate goals to create an environment promoting health and health equity.

The unfolded protein response (UPR) allows cells to adjust secretory pathway capacity according to need. Ire1, the endoplasmic reticulum (ER) stress sensor and central activator of the UPR is conserved from the budding yeast Saccharomyces cerevisiae to humans. Under ER stress conditions, Ire1 clusters into foci that enable optimal UPR activation. To discover factors that affect Ire1 clustering, we performed a high-content screen using a whole-genome yeast mutant library expressing Ire1–mCherry. We imaged the strains following UPR induction and found 154 strains that displayed alterations in Ire1 clustering. The hits were enriched for iron and heme effectors and binding proteins. By performing pharmacological depletion and repletion, we confirmed that iron (Fe3+) affects UPR activation in both yeast and human cells. We suggest that Ire1 clustering propensity depends on membrane composition, which is governed by heme-dependent biosynthesis of sterols. Our findings highlight the diverse cellular functions that feed into the UPR and emphasize the cross-talk between organelles required to concertedly maintain homeostasis., Highlighted Article: To respond to folding stress in the ER, cells activate the conserved sensor Ire1. We show that iron is required for optimal Ire1 activation and suggest this is because iron is required for ergosterol biosynthesis.

Background & Aims Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. Methods We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. Results Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene ( Stat3 ). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. Conclusion In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.

The unfolded protein response (UPR) governs homeostasis of both luminal content and membrane of the endoplasmic reticulum (ER). In Molecular Cell, Halbleib et al. identified how a twist in the juxta-membrane amphipathic helix of the UPR transducer Ire1 in yeast is essential for responding to both proteostatic and lipostatic ER stress.