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Article ; Online: Expression of the Z Variant of α1-Antitrypsin Suppresses Hepatic Cholesterol Biosynthesis in Transgenic Zebrafish

Connie Fung / Brendan Wilding / Ralf B. Schittenhelm / Robert J. Bryson-Richardson / Phillip I. Bird

International Journal of Molecular Sciences, Vol 24, Iss 2475, p

2023 Volume 2475

Abstract: Individuals homozygous for the Pi*Z allele of SERPINA1 (ZAAT) are susceptible to lung disease due to insufficient α1-antitrypsin secretion into the circulation and may develop liver disease due to compromised protein folding that leads to inclusion body ... ...

Abstract	Individuals homozygous for the Pi*Z allele of SERPINA1 (ZAAT) are susceptible to lung disease due to insufficient α1-antitrypsin secretion into the circulation and may develop liver disease due to compromised protein folding that leads to inclusion body formation in the endoplasmic reticulum (ER) of hepatocytes. Transgenic zebrafish expressing human ZAAT show no signs of hepatic accumulation despite displaying serum insufficiency, suggesting the defect in ZAAT secretion occurs independently of its tendency to form inclusion bodies. In this study, proteomic, transcriptomic, and biochemical analysis provided evidence of suppressed Srebp2-mediated cholesterol biosynthesis in the liver of ZAAT-expressing zebrafish. To investigate the basis for this perturbation, CRISPR/Cas9 gene editing was used to manipulate ER protein quality control factors. Mutation of erlec1 resulted in a further suppression in the cholesterol biosynthesis pathway, confirming a role for this ER lectin in targeting misfolded ZAAT for ER-associated degradation (ERAD). Mutation of the two ER mannosidase homologs enhanced ZAAT secretion without inducing hepatic accumulation. These insights into hepatic ZAAT processing suggest potential therapeutic targets to improve secretion and alleviate serum insufficiency in this form of the α1-antitrypsin disease.
Keywords	α-1-antitrypsin ; SERPINA1 ; zebrafish ; liver ; ERAD ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2023-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Exploring the selectivity and engineering potential of an NRPS condensation domain involved in the biosynthesis of the thermophilic siderophore fuscachelin

Y. T. Candace Ho / Thierry Izoré / Joe A. Kaczmarski / Edward Marschall / Minuri S. Ratnayake / Julien Tailhades / David L. Steer / Ralf B. Schittenhelm / Manuela Tosin / Colin J. Jackson / Max J. Cryle

Frontiers in Catalysis, Vol

2023 Volume 3

Abstract: In nonribosomal peptide synthesis, condensation (C) domains are key catalytic domains that most commonly link carrier protein bound substrates to form peptides or depsipeptides. While adenylation domains have been well characterized due to their role in ... ...

Abstract	In nonribosomal peptide synthesis, condensation (C) domains are key catalytic domains that most commonly link carrier protein bound substrates to form peptides or depsipeptides. While adenylation domains have been well characterized due to their role in the selection of monomers and hence as gate keepers in nonribosomal peptide biosynthesis, C-domains have been the subject of debate as they do not have apparent “A-domain like” side chain selectivity for their acceptor substrates. To probe the selectivity and specificity of C-domains, here we report our biochemical and structural characterization of the C3-domain from the biosynthesis of the siderophore fusachelin. Our results show that this C-domain is not broadly flexible for monomers bearing significantly alternated side chains or backbones, which suggests there can be a need to consider C-domain specificity for acceptor substrates when undertaking NRPS engineering.
Keywords	nonribosomal peptide (NRP) ; biosynthesis ; condensation domain ; peptidyl carrier protein ; biocatalysis ; depsipeptide ; Chemistry ; QD1-999
Subject code	540
Language	English
Publishing date	2023-06-01T00:00:00Z
Publisher	Frontiers Media S.A.
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Data-Independent-Acquisition-Based Proteomic Approach towards Understanding the Acclimation Strategy of Oleaginous Microalga Microchloropsis gaditana CCMP526 in Hypersaline Conditions

Anbarasu Karthikaichamy / John Beardall / Ross Coppel / Santosh Noronha / Dieter Bulach / Ralf B. Schittenhelm / Sanjeeva Srivastava

ACS Omega, Vol 6, Iss 34, Pp 22151-

2021 Volume 22164

Keywords	Chemistry ; QD1-999
Language	English
Publishing date	2021-08-01T00:00:00Z
Publisher	American Chemical Society
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages.

Zhi Ying Kho / Mohammad A K Azad / Mei-Ling Han / Yan Zhu / Cheng Huang / Ralf B Schittenhelm / Thomas Naderer / Tony Velkov / Joel Selkrig / Qi Tony Zhou / Jian Li

PLoS Pathogens, Vol 18, Iss 3, p e

2022 Volume 1010308

Abstract: The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial ... ...

Abstract	The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages

Zhi Ying Kho / Mohammad A. K. Azad / Mei-Ling Han / Yan Zhu / Cheng Huang / Ralf B. Schittenhelm / Thomas Naderer / Tony Velkov / Joel Selkrig / Qi (Tony) Zhou / Jian Li

PLoS Pathogens, Vol 18, Iss

2022 Volume 3

Abstract	The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections. Author summary Bacterial response towards antibiotics is sensitive to the surrounding environment; however, how the host immune microenvironment affects the response of opportunistic pathogen Acinetobacter baumannii towards polymyxins remains largely unexplored. In this study, we established a host-pathogen-antibiotic tripartite in vitro model to examine the complex tripartite molecular interplay from both bacteria and macrophages perspectives, mimicking the physiological infection-treatment condition. This is the first comprehensive proteomics dataset for A. baumannii interacting with host cells, with an extraordinary coverage (i.e., 50%) of A. baumannii proteome even in infection conditions. For the first time, we report that macrophages and polymyxins utilize complementary mechanisms to disarm several A. baumannii stress tolerance strategies to persist in the macrophages. Our host-pathogen-antibiotic mechanistic study and the discovery of potential druggable targets (e.g., bacterial stringent stress response regulator SpoT) represent a significant advance, paving way to antibiotic optimization and drug discovery to tackle multidrug-resistant bacterial infections.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage

Anja Greule / Thierry Izoré / Daniel Machell / Mathias H. Hansen / Melanie Schoppet / James J. De Voss / Louise K. Charkoudian / Ralf B. Schittenhelm / Jeffrey R. Harmer / Max J. Cryle

Frontiers in Chemistry, Vol

2022 Volume 10

Abstract: Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate–ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within ... ...

Abstract	Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate–ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid auto-oxidation and retain the axial cysteine ligand in the deprotonated—and thus highly acidic—thiolate form. While little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site. Indeed, very few structures to date contain an alternate heme orientation, of which two are OxyA homologs from glycopeptide antibiotic (GPA) biosynthesis. Given the apparent preference for the unusual heme orientation shown by OxyA enzymes, we investigated the OxyA homolog from kistamicin biosynthesis (OxyAkis), which is an atypical GPA. We determined that OxyAkis is highly sensitive to oxidative damage by peroxide, with both UV and EPR measurements showing rapid bleaching of the heme signal. We determined the structure of OxyAkis and found a mixed population of heme orientations present in this enzyme. Our analysis further revealed the possible modification of the heme moiety, which was only present in samples where the alternate heme orientation was present in the protein. These results suggest that the typical heme orientation in cytochrome P450s can help prevent potential damage to the heme—and hence deactivation of the enzyme—during P450 catalysis. It also suggests that some P450 enzymes involved in GPA biosynthesis may be especially prone to oxidative damage due to the heme orientation found in their active sites.
Keywords	cytochrome P450 ; glycopeptide antibiotic biosynthesis ; kistamicin ; heme ; biosynthesis ; Chemistry ; QD1-999
Subject code	500 ; 612
Language	English
Publishing date	2022-04-01T00:00:00Z
Publisher	Frontiers Media S.A.
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

Hirokazu Okada / Ralf B Schittenhelm / Anna Straessle / Ernst Hafen

PLoS ONE, Vol 10, Iss 3, p e

2015 Volume 0113902

Abstract: The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological ... ...

Abstract	The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2015-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability

Eric Mandela / Christopher J Stubenrauch / David Ryoo / Hyea Hwang / Eli J Cohen / Von L Torres / Pankaj Deo / Chaille T Webb / Cheng Huang / Ralf B Schittenhelm / Morgan Beeby / JC Gumbart / Trevor Lithgow / Iain D Hay

eLife, Vol

2022 Volume 11

Abstract: The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and ... ...

Abstract	The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation
Keywords	periplasm ; cell envelope ; Lpp ; peptidoglycan ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: An Integrated Multi-Omic Network Analysis Identifies Seizure-Associated Dysregulated Pathways in the GAERS Model of Absence Epilepsy

Anna Harutyunyan / Debbie Chong / Rui Li / Anup D. Shah / Zahra Ali / Cheng Huang / Christopher K. Barlow / Piero Perucca / Terence J. O’Brien / Nigel C. Jones / Ralf B. Schittenhelm / Alison Anderson / Pablo M. Casillas-Espinosa

International Journal of Molecular Sciences, Vol 23, Iss 6063, p

2022 Volume 6063

Abstract: Absence epilepsy syndromes are part of the genetic generalized epilepsies, the pathogenesis of which remains poorly understood, although a polygenic architecture is presumed. Current focus on single molecule or gene identification to elucidate ... ...

Abstract	Absence epilepsy syndromes are part of the genetic generalized epilepsies, the pathogenesis of which remains poorly understood, although a polygenic architecture is presumed. Current focus on single molecule or gene identification to elucidate epileptogenic drivers is unable to fully capture the complex dysfunctional interactions occurring at a genetic/proteomic/metabolomic level. Here, we employ a multi-omic, network-based approach to characterize the molecular signature associated with absence epilepsy-like phenotype seen in a well validated rat model of genetic generalized epilepsy with absence seizures. Electroencephalographic and behavioral data was collected from Genetic Absence Epilepsy Rats from Strasbourg (GAERS, n = 6) and non-epileptic controls (NEC, n = 6), followed by proteomic and metabolomic profiling of the cortical and thalamic tissue of rats from both groups. The general framework of weighted correlation network analysis (WGCNA) was used to identify groups of highly correlated proteins and metabolites, which were then functionally annotated through joint pathway enrichment analysis. In both brain regions a large protein-metabolite module was found to be highly associated with the GAERS strain, absence seizures and associated anxiety and depressive-like phenotype. Quantitative pathway analysis indicated enrichment in oxidative pathways and a downregulation of the lysine degradation pathway in both brain regions. GSTM1 and ALDH2 were identified as central regulatory hubs of the seizure-associated module in the somatosensory cortex and thalamus, respectively. These enzymes are involved in lysine degradation and play important roles in maintaining oxidative balance. We conclude that the dysregulated pathways identified in the seizure-associated module may be involved in the aetiology and maintenance of absence seizure activity. This dysregulated activity could potentially be modulated by targeting one or both central regulatory hubs.
Keywords	absence epilepsy ; ALDH2 ; GAERS ; GSTM1 ; lysine degradation ; metabolomics ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	616
Language	English
Publishing date	2022-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

Rachael Emily Turner / Paul F Harrison / Angavai Swaminathan / Calvin A Kraupner-Taylor / Belinda J Goldie / Michael See / Amanda L Peterson / Ralf B Schittenhelm / David R Powell / Darren J Creek / Bernhard Dichtl / Traude H Beilharz

eLife, Vol

2021 Volume 10

Abstract: Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3’ end formation factors, which revealed 3’UTR lengthening in response to ... ...

Abstract	Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3’ end formation factors, which revealed 3’UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3’UTRs. We show that the anti-cancer drug cordycepin, 3’ deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3’ end processing and controlled by the availability of 3’ end factors, nucleotide levels and chromatin landscape.
Keywords	alternative polyadenylation ; transcription elongation ; cleavage ; polyadenylation ; cordycepin ; mycophenolic acid ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2021-07-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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