Publications by authors named "Ralf B Schittenhelm"

59 Publications

crisscrosslinkeR: identification and visualization of protein-RNA and protein-protein interactions from crosslinking mass spectrometry.

Bioinformatics 2020 Dec 21. Epub 2020 Dec 21.

Department of Biochemistry and Molecular Biology, Monash Proteomics & Metabolomics Facility.

Summary: Unbiased detection of protein-protein and protein-RNA interactions within ribonucleoprotein complexes are enabled through crosslinking followed by mass spectrometry. Yet, different methods detect different types of molecular interactions and therefore require the usage of different software packages with limited compatibility. We present crisscrosslinkeR, an R package that maps both protein-protein and protein-RNA interactions detected by different types of approaches for crosslinking with mass spectrometry. crisscrosslinkeR produces output files that are compatible with visualization using popular software packages for the generation of publication-quality figures.

Availability And Implementation: crisscrosslinkeR is a free and open-source package, available through GitHub: github.com/egmg726/crisscrosslinker.

Supplementary Information: Workflows are available at https://egmg726.github.io/crisscrosslinker/.
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http://dx.doi.org/10.1093/bioinformatics/btaa1043DOI Listing
December 2020

Perilipin 5 S155 phosphorylation by PKA is required for the control of hepatic lipid metabolism and glycemic control.

J Lipid Res 2021 Jan 5;62:100016. Epub 2021 Jan 5.

Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia. Electronic address:

Perilipin 5 (PLIN5) is a lipid-droplet-associated protein that coordinates intracellular lipolysis in highly oxidative tissues and is thought to regulate lipid metabolism in response to phosphorylation by protein kinase A (PKA). We sought to identify PKA phosphorylation sites in PLIN5 and assess their functional relevance in cultured cells and the livers of mice. We detected phosphorylation on S155 and identified S155 as a functionally important site for lipid metabolism. Expression of phosphorylation-defective PLIN5 S155A in Plin5 null cells resulted in decreased rates of lipolysis and triglyceride-derived fatty acid oxidation. FLIM-FRET analysis of protein-protein interactions showed that PLIN5 S155 phosphorylation regulates PLIN5 interaction with adipose triglyceride lipase at the lipid droplet, but not with α-β hydrolase domain-containing 5. Re-expression of PLIN5 S155A in the liver of Plin5 liver-specific null mice reduced lipolysis compared with wild-type PLIN5 re-expression, but was not associated with other changes in hepatic lipid metabolism. Furthermore, glycemic control was impaired in mice with expression of PLIN5 S155A compared with mice expressing PLIN5. Together, these studies demonstrate that PLIN5 S155 is required for PKA-mediated lipolysis and builds on the body of evidence demonstrating a critical role for PLIN5 in coordinating lipid and glucose metabolism.
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http://dx.doi.org/10.1194/jlr.RA120001126DOI Listing
January 2021

TINC- A Method to Dissect Regulatory Complexes at Single-Locus Resolution- Reveals an Extensive Protein Complex at the Nanog Promoter.

Stem Cell Reports 2020 12;15(6):1246-1259

Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.
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http://dx.doi.org/10.1016/j.stemcr.2020.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724517PMC
December 2020

RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells.

Elife 2020 12 2;9. Epub 2020 Dec 2.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8 T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.
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http://dx.doi.org/10.7554/eLife.63452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710356PMC
December 2020

The RSC (Remodels the Structure of Chromatin) complex of Candida albicans shows compositional divergence with distinct roles in regulating pathogenic traits.

PLoS Genet 2020 11 5;16(11):e1009071. Epub 2020 Nov 5.

Department of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai, India.

Regulation of gene expression programs is crucial for the survival of microbial pathogens in host environments and for their ability to cause disease. Here we investigated the epigenetic regulator RSC (Remodels the Structure of Chromatin) in the most prevalent human fungal pathogen Candida albicans. Biochemical analysis showed that CaRSC comprises 13 subunits and contains two novel non-essential members, which we named Nri1 and Nri2 (Novel RSC Interactors) that are exclusive to the CTG clade of Saccharomycotina. Genetic analysis showed distinct essentiality of C. albicans RSC subunits compared to model fungal species suggesting functional and structural divergence of RSC functions in this fungal pathogen. Transcriptomic and proteomic profiling of a conditional mutant of the essential catalytic subunit gene STH1 demonstrated global roles of RSC in C. albicans biology, with the majority of growth-related processes affected, as well as mis-regulation of genes involved in morphotype switching, host-pathogen interaction and adaptive fitness. We further assessed the functions of non-essential CaRSC subunits, showing that the novel subunit Nri1 and the bromodomain subunit Rsc4 play roles in filamentation and stress responses; and also interacted at the genetic level to regulate cell viability. Consistent with these roles, Rsc4 is required for full virulence of C. albicans in the murine model of systemic infection. Taken together, our data builds the first comprehensive study of the composition and roles of RSC in C. albicans, showing both conserved and distinct features compared to model fungal systems. The study illuminates how C. albicans uses RSC-dependent transcriptional regulation to respond to environmental signals and drive survival fitness and virulence in mammals.
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http://dx.doi.org/10.1371/journal.pgen.1009071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671503PMC
November 2020

Behavioral, axonal, and proteomic alterations following repeated mild traumatic brain injury: Novel insights using a clinically relevant rat model.

Neurobiol Dis 2021 Jan 27;148:105151. Epub 2020 Oct 27.

Department of Physiology, Anatomy and Microbiology, School of Life Sciences, La Trobe University, Melbourne, VIC 3086, Australia; Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC 3004, Australia. Electronic address:

A history of mild traumatic brain injury (mTBI) is linked to a number of chronic neurological conditions, however there is still much unknown about the underlying mechanisms. To provide new insights, this study used a clinically relevant model of repeated mTBI in rats to characterize the acute and chronic neuropathological and neurobehavioral consequences of these injuries. Rats were given four sham-injuries or four mTBIs and allocated to 7-day or 3.5-months post-injury recovery groups. Behavioral analysis assessed sensorimotor function, locomotion, anxiety, and spatial memory. Neuropathological analysis included serum quantification of neurofilament light (NfL), mass spectrometry of the hippocampal proteome, and ex vivo magnetic resonance imaging (MRI). Repeated mTBI rats had evidence of acute cognitive deficits and prolonged sensorimotor impairments. Serum NfL was elevated at 7 days post injury, with levels correlating with sensorimotor deficits; however, no NfL differences were observed at 3.5 months. Several hippocampal proteins were altered by repeated mTBI, including those associated with energy metabolism, neuroinflammation, and impaired neurogenic capacity. Diffusion MRI analysis at 3.5 months found widespread reductions in white matter integrity. Taken together, these findings provide novel insights into the nature and progression of repeated mTBI neuropathology that may underlie lingering or chronic neurobehavioral deficits.
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http://dx.doi.org/10.1016/j.nbd.2020.105151DOI Listing
January 2021

Immune molecular profiling of a multiresistant primary prostate cancer with a neuroendocrine-like phenotype: a case report.

BMC Urol 2020 Oct 28;20(1):171. Epub 2020 Oct 28.

Tumor Suppression Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia.

Background: Understanding the drivers of recurrence in aggressive prostate cancer requires detailed molecular and genomic understanding in order to aid therapeutic interventions. We provide here a case report of histological, transcriptional, proteomic, immunological, and genomic features in a longitudinal study of multiple biopsies from diagnosis, through treatment, and subsequent recurrence.

Case Presentation: Here we present a case study of a male in 70 s with high-grade clinically-localised acinar adenocarcinoma treated with definitive hormone therapy and radiotherapy. The patient progressed rapidly with rising PSA and succumbed without metastasis 52 months after diagnosis. We identified the expression of canonical histological markers of neuroendocrine PC (NEPC) including synaptophysin, neuron-specific enolase and thyroid transcription factor 1, as well as intact AR expression, in the recurrent disease only. The resistant disease was also marked by an extremely low immune infiltrate, extensive genomic chromosomal aberrations, and overactivity in molecular hallmarks of NEPC disease including Aurora kinase and E2F, as well as novel alterations in the cMYB pathway. We also observed that responses to both primary treatments (high dose-rate brachytherapy and androgen deprivation therapies) were consistent with known optimal responses-ruling out treatment inefficacy as a factor in relapse.

Conclusions: These data provide novel insights into a case of locally recurrent aggressive prostate cancer harbouring NEPC pathology, in the absence of detected metastasis.
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http://dx.doi.org/10.1186/s12894-020-00738-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592533PMC
October 2020

Spliced Peptides and Cytokine-Driven Changes in the Immunopeptidome of Melanoma.

Cancer Immunol Res 2020 10 16;8(10):1322-1334. Epub 2020 Sep 16.

Cancer Immunobiology, Olivia Newton-John Cancer Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.

Antigen recognition by CD8 T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of noncontiguous regions of proteins to form novel peptide antigens. Here, we used high-resolution mass spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of IFNγ. In total, we identified more than 60,000 peptides from a single patient-derived cell line (LM-MEL-44) and demonstrated that IFNγ induced changes in the peptidome, with an overlap of only approximately 50% between basal and treated cells. Around 6% to 8% of the peptides were identified as -spliced peptides, and 2,213 peptides (1,827 linear and 386 -spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive- and IFNγ-induced peptidome. We next examined additional HLA-matched patient-derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides was conserved between individual patient tumors, drawing on data amassing to more than 100,000 peptide sequences. Several of these peptides showed immunogenicity across multiple patients with melanoma. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major class of tumor antigens.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0894DOI Listing
October 2020

SMOC1 is a glucose-responsive hepatokine and therapeutic target for glycemic control.

Sci Transl Med 2020 09;12(559)

Department of Physiology, University of Melbourne, Melbourne, VIC 3010, Australia.

Intertissue communication is a fundamental feature of metabolic regulation, and the liver is central to this process. We have identified sparc-related modular calcium-binding protein 1 (SMOC1) as a glucose-responsive hepatokine and regulator of glucose homeostasis. Acute intraperitoneal administration of SMOC1 improved glycemic control and insulin sensitivity in mice without changes in insulin secretion. SMOC1 exerted its favorable glycemic effects by inhibiting adenosine 3',5'-cyclic monophosphate (cAMP)-cAMP-dependent protein kinase (PKA)-cAMP response element-binding protein (CREB) signaling in the liver, leading to decreased gluconeogenic gene expression and suppression of hepatic glucose output. Overexpression of SMOC1 in the liver or once-weekly intraperitoneal injections of a stabilized SMOC1-FC fusion protein induced durable improvements in glucose tolerance and insulin sensitivity in / mice, without adverse effects on adiposity, liver histopathology, or inflammation. Furthermore, circulating SMOC1 correlated with hepatic and systemic insulin sensitivity and was decreased in obese, insulin-resistant humans. Together, these findings identify SMOC1 as a potential pharmacological target for the management of glycemic control in type 2 diabetes.
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http://dx.doi.org/10.1126/scitranslmed.aaz8048DOI Listing
September 2020

Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation.

Nat Microbiol 2020 11 17;5(11):1418-1427. Epub 2020 Aug 17.

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

Sensing of microbes activates the innate immune system, depending on functional mitochondria. However, pathogenic bacteria inhibit mitochondrial activity by delivering toxins via outer membrane vesicles (OMVs). How macrophages respond to pathogenic microbes that target mitochondria remains unclear. Here, we show that macrophages exposed to OMVs from Neisseria gonorrhoeae, uropathogenic Escherichia coli and Pseudomonas aeruginosa induce mitochondrial apoptosis and NLRP3 inflammasome activation. OMVs and toxins that cause mitochondrial dysfunction trigger inhibition of host protein synthesis, which depletes the unstable BCL-2 family member MCL-1 and induces BAK-dependent mitochondrial apoptosis. In parallel with caspase-11-mediated pyroptosis, mitochondrial apoptosis and potassium ion efflux activate the NLRP3 inflammasome after OMV exposure in vitro. Importantly, in the in vivo setting, the activation and release of interleukin-1β in response to N. gonorrhoeae OMVs is regulated by mitochondrial apoptosis. Our data highlight how innate immune cells sense infections by monitoring mitochondrial health.
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http://dx.doi.org/10.1038/s41564-020-0773-2DOI Listing
November 2020

Redesign of Substrate Selection in Glycopeptide Antibiotic Biosynthesis Enables Effective Formation of Alternate Peptide Backbones.

ACS Chem Biol 2020 09 31;15(9):2444-2455. Epub 2020 Aug 31.

The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

Nonribosomal peptide synthesis is capable of utilizing a wide range of amino acid residues due to the selectivity of adenylation (A)-domains. Changing the selectivity of A-domains could lead to new bioactive nonribosomal peptides, although remodeling efforts of A-domains are often unsuccessful. Here, we explored and successfully reengineered the specificity of the module 3 A-domain from glycopeptide antibiotic biosynthesis to change the incorporation of 3,5-dihydroxyphenylglycine into 4-hydroxyphenylglycine. These engineered A-domains remain selective in a functioning peptide assembly line even under substrate competition conditions and indicate a possible application of these for the future redesign of GPA biosynthesis.
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http://dx.doi.org/10.1021/acschembio.0c00435DOI Listing
September 2020

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution.

Nat Commun 2020 06 9;11(1):2894. Epub 2020 Jun 9.

Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.
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http://dx.doi.org/10.1038/s41467-020-16568-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283339PMC
June 2020

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F-0 in Mycobacteria.

mSystems 2020 May 19;5(3). Epub 2020 May 19.

School of Biological Sciences, Monash University, Clayton, VIC, Australia

F is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F biosynthesis in mycobacterial cells. We show that, in whole cells of , phosphoenolpyruvate rather than 2-phospholactate stimulates F biosynthesis. Analysis of F biosynthesis intermediates present in cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F-0. To determine the structural basis of dehydro-F-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F.
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http://dx.doi.org/10.1128/mSystems.00389-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253369PMC
May 2020

A Natural Peptide Antigen within the Plasmodium Ribosomal Protein RPL6 Confers Liver T Cell-Mediated Immunity against Malaria in Mice.

Cell Host Microbe 2020 06 11;27(6):950-962.e7. Epub 2020 May 11.

Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Liver-resident memory CD8 T (T) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form T cells. Here, we identify PbRPL6, a H2-K-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver T cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A02:01-restricted epitope in P. falciparum RPL6.
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http://dx.doi.org/10.1016/j.chom.2020.04.010DOI Listing
June 2020

Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics.

FEBS J 2021 Jan 30;288(2):507-529. Epub 2020 May 30.

Department of Biochemistry and Molecular Biology, The Monash Biomedicine Discovery Institute, Monash University, Clayton, Australia.

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.
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http://dx.doi.org/10.1111/febs.15350DOI Listing
January 2021

Interaction of Plasmodium falciparum casein kinase 1 with components of host cell protein trafficking machinery.

IUBMB Life 2020 Jun 1;72(6):1243-1249. Epub 2020 May 1.

Infection and Immunity Program and Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

A pool of Plasmodium falciparum casein kinase 1 (PfCK1) has been shown to localize to the host red blood cell (RBC) membrane and be secreted to the extracellular medium during trophozoite stage of development. We attempted to identify mechanisms for secretion of PfCK1 and its appearance on the RBC membrane. We found that two host proteins with established functions in membrane trafficking in higher eukaryotes, GTPase-activating protein and Vps9 domain-containing protein 1 (GAPVD1), and Sorting nexin 22, consistently co-purify with PfCK1, suggesting that the parasite utilizes trafficking pathways previously thought to be inactive in RBCs. Furthermore, reciprocal immunoprecipitation experiments with GAPVD1 identified parasite proteins suggestive of a protein recycling pathway hitherto only described in higher eukaryotes. Thus, we have identified components of a trafficking pathway involving parasite proteins that act in concert with host proteins, and which we hypothesize mediates trafficking of PfCK1 to the RBC during infection.
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http://dx.doi.org/10.1002/iub.2294DOI Listing
June 2020

A Chemoenzymatic Approach to the Synthesis of Glycopeptide Antibiotic Analogues.

Angew Chem Int Ed Engl 2020 06 11;59(27):10899-10903. Epub 2020 May 11.

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, 3800, Australia.

Glycopeptide antibiotics (GPAs) are important antibiotics that are highly challenging to synthesise due to their unique and heavily crosslinked structure. Given this, the synthetic production and diversification of this key compound class remains impractical. Furthermore, the possibility of biosynthetic reengineering of GPAs is not yet feasible since the selectivity of the biosynthetic crosslinking enzymes for altered substrates is largely unknown. We show that combining peptide synthesis with enzymatic cyclisation enables the formation of novel examples of GPAs and provides an indication of the utility of these crucial enzymes. By accessing the biosynthetic process in vitro, we identified peptide modifications that are enzymatically tolerated and can also reveal the mechanistic basis for substrate intolerance where present. Using this approach, we next specifically activated modified residues within GPAs for functionalisation at previously inaccessible positions, thereby offering the possibility of late-stage chemical functionalisation after GPA cyclisation is complete.
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http://dx.doi.org/10.1002/anie.202003726DOI Listing
June 2020

Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2.

J Biol Chem 2020 05 2;295(19):6518-6531. Epub 2020 Apr 2.

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton 3800, Victoria, Australia

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.
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http://dx.doi.org/10.1074/jbc.RA119.012026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212651PMC
May 2020

A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics.

Chem Sci 2019 Nov 29;10(41):9466-9482. Epub 2019 Aug 29.

The Monash Biomedicine Discovery Institute , Department of Biochemistry and Molecular Biology , Monash University , Clayton , Victoria 3800 , Australia . Email:

Non-ribosomal peptide biosynthesis produces highly diverse natural products through a complex cascade of enzymatic reactions that together function with high selectivity to produce bioactive peptides. The modification of non-ribosomal peptide synthetase (NRPS)-bound amino acids can introduce significant structural diversity into these peptides and has exciting potential for biosynthetic redesign. However, the control mechanisms ensuring selective modification of specific residues during NRPS biosynthesis have previously been unclear. Here, we have characterised the incorporation of the non-proteinogenic amino acid 3-chloro-β-hydroxytyrosine during glycopeptide antibiotic (GPA) biosynthesis. Our results demonstrate that the modification of this residue by -acting enzymes is controlled by the selectivity of the upstream condensation domain responsible for peptide synthesis. A proofreading thioesterase works together with this process to ensure that effective peptide biosynthesis proceeds even when the selectivity of key amino acid activation domains within the NRPS is low. Furthermore, the exchange of condensation domains with altered amino acid specificities allows the modification of such residues within NRPS biosynthesis to be controlled, which will doubtless prove important for reengineering of these assembly lines. Taken together, our results indicate the importance of the complex interplay of NRPS domains and -acting enzymes to ensure effective GPA biosynthesis, and in doing so reveals a process that is mechanistically comparable to the hydrolytic proofreading function of tRNA synthetases in ribosomal protein synthesis.
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http://dx.doi.org/10.1039/c9sc03678dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993612PMC
November 2019

Exploring the Tetracyclization of Teicoplanin Precursor Peptides through Chemoenzymatic Synthesis.

J Org Chem 2020 02 11;85(3):1537-1547. Epub 2019 Dec 11.

The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology , Monash University , Clayton , Victoria 3800 , Australia.

The glycopeptide antibiotics (GPAs) serve as an important example of the interplay of two powerful enzymatic classes in secondary metabolism: the coupling of nonribosomal peptide synthesis with oxidative aromatic cross-linking performed by cytochrome P450 enzymes. This interplay is responsible for the generation of the highly cross-linked peptide aglycone at the core of this compound class that is required for antibiotic activity and, as such, serves as an important point for the exploration of chemoenzymatic routes to understand the selectivity and mechanism of this complex cascade. Here, we demonstrate the effective reconstitution of enzymatic tetracyclization of synthetic teicoplanin-derived heptapeptides and furthermore discern the importance of the OxyE enzyme in maintaining effective cyclization of such peptides bearing 3,5-dihydroxyphenylglycine residues at position 3 in their structures. These results demonstrate the value of chemically synthesized probes for the elucidation of the enzyme mechanism underpinning the complex process of GPA cyclization and furthermore show the utility of the technique for probing the cyclization of non-natural GPA peptides by these powerful biosynthetic enzymes.
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http://dx.doi.org/10.1021/acs.joc.9b02640DOI Listing
February 2020

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

ACS Chem Biol 2019 12 10;14(12):2932-2941. Epub 2019 Dec 10.

The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology , Monash University , Clayton , Victoria 3800 , Australia.

β-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the utility and scope of the unusual nonheme diiron monooxygenase CmlA from chloramphenicol biosynthesis for the β-hydroxylation of a diverse range of carrier protein bound substrates by adapting this enzyme as a non-native -acting enzyme within NRPS-mediated GPA biosynthesis. The results from our study show that CmlA has a broad substrate specificity for modified phenylalanine/tyrosine residues as substrates and can be used in a practical strategy to functionally cross complement compatible NRPS biosynthesis pathways .
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http://dx.doi.org/10.1021/acschembio.9b00862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929969PMC
December 2019

Allelic association with ankylosing spondylitis fails to correlate with human leukocyte antigen B27 homodimer formation.

J Biol Chem 2019 12 18;294(52):20185-20195. Epub 2019 Nov 18.

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia

Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition toward ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis. Here, we examined the ability of the eight most prevalent HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09) to form homodimers. We observed that HLA-B*27:03, a disease-associated HLA-B27 subtype, showed a significantly reduced ability to form homodimers compared with all other allotypes, including the non-disease-associated/protective allotypes HLA-B*27:06 and HLA-B*27:09. We used X-ray crystallography and site-directed mutagenesis to unravel the molecular and structural mechanisms in HLA-B*27:03 that are responsible for its compromised ability to form homodimers. We show that polymorphism at position 59, which differentiates HLA-B*27:03 from all other allotypes, is responsible for its compromised ability to form homodimers. Indeed, histidine 59 in HLA-B*27:03 leads to a series of local conformational changes that act in concert to reduce the accessibility of the nearby cysteine 67, an essential amino acid residue for the formation of HLA-B27 homodimers. Considered together, the ability of both protective and disease-associated HLA-B27 allotypes to form homodimers and the failure of HLA-B*27:03 to form homodimers challenge the role of HLA-B27 homodimers in AS pathoetiology. Rather, this work implicates other features, such as peptide binding and antigen presentation, as pivotal mechanisms for disease pathogenesis.
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http://dx.doi.org/10.1074/jbc.RA119.010257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937573PMC
December 2019

LFQ-Analyst: An Easy-To-Use Interactive Web Platform To Analyze and Visualize Label-Free Proteomics Data Preprocessed with MaxQuant.

J Proteome Res 2020 01 8;19(1):204-211. Epub 2019 Nov 8.

Relative label-free quantification (LFQ) of shotgun proteomics data using precursor (MS1) signal intensities is one of the most commonly used applications to comprehensively and globally quantify proteins across biological samples and conditions. Due to the popularity of this technique, several software packages, such as the popular software suite MaxQuant, have been developed to extract, analyze, and compare spectral features and to report quantitative information of peptides, proteins, and even post-translationally modified sites. However, there is still a lack of accessible tools for the interpretation and downstream statistical analysis of these complex data sets, in particular for researchers and biologists with no or only limited experience in proteomics, bioinformatics, and statistics. We have therefore created LFQ-Analyst, which is an easy-to-use, interactive web application developed to perform differential expression analysis with "one click" and to visualize label-free quantitative proteomic data sets preprocessed with MaxQuant. LFQ-Analyst provides a wealth of user-analytic features and offers numerous publication-quality result graphics to facilitate statistical and exploratory analysis of label-free quantitative data sets. LFQ-Analyst, including an in-depth user manual, is freely available at https://bioinformatics.erc.monash.edu/apps/LFQ-Analyst .
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http://dx.doi.org/10.1021/acs.jproteome.9b00496DOI Listing
January 2020

Enzymatic Cascade To Evaluate the Tricyclization of Glycopeptide Antibiotic Precursor Peptides as a Prequel to Biosynthetic Redesign.

Org Lett 2019 11 11;21(21):8635-8640. Epub 2019 Oct 11.

The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology , Monash University , Clayton , Victoria 3800 , Australia.

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.
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http://dx.doi.org/10.1021/acs.orglett.9b03245DOI Listing
November 2019

Ligand-dependent spatiotemporal signaling profiles of the μ-opioid receptor are controlled by distinct protein-interaction networks.

J Biol Chem 2019 11 12;294(44):16198-16213. Epub 2019 Sep 12.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia

Ligand-dependent differences in the regulation and internalization of the μ-opioid receptor (MOR) have been linked to the severity of adverse effects that limit opiate use in pain management. MOR activation by morphine or [d-Ala,-MePhe, Gly-ol]enkephalin (DAMGO) causes differences in spatiotemporal signaling dependent on MOR distribution at the plasma membrane. Morphine stimulation of MOR activates a Gα-Gβγ-protein kinase C (PKC) α phosphorylation pathway that limits MOR distribution and is associated with a sustained increase in cytosolic extracellular signal-regulated kinase (ERK) activity. In contrast, DAMGO causes a redistribution of the MOR at the plasma membrane (before receptor internalization) that facilitates transient activation of cytosolic and nuclear ERK. Here, we used proximity biotinylation proteomics to dissect the different protein-interaction networks that underlie the spatiotemporal signaling of morphine and DAMGO. We found that DAMGO, but not morphine, activates Ras-related C3 botulinum toxin substrate 1 (Rac1). Both Rac1 and nuclear ERK activity depended on the scaffolding proteins IQ motif-containing GTPase-activating protein-1 (IQGAP1) and Crk-like (CRKL) protein. In contrast, morphine increased the proximity of the MOR to desmosomal proteins, which form specialized and highly-ordered membrane domains. Knockdown of two desmosomal proteins, junction plakoglobin or desmocolin-1, switched the morphine spatiotemporal signaling profile to mimic that of DAMGO, resulting in a transient increase in nuclear ERK activity. The identification of the MOR-interaction networks that control differential spatiotemporal signaling reported here is an important step toward understanding how signal compartmentalization contributes to opioid-induced responses, including anti-nociception and the development of tolerance and dependence.
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http://dx.doi.org/10.1074/jbc.RA119.008685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827304PMC
November 2019

Atmospheric carbon monoxide oxidation is a widespread mechanism supporting microbial survival.

ISME J 2019 11 29;13(11):2868-2881. Epub 2019 Jul 29.

School of Biological Sciences, Monash University, Clayton, VIC, 3800, Australia.

Carbon monoxide (CO) is a ubiquitous atmospheric trace gas produced by natural and anthropogenic sources. Some aerobic bacteria can oxidize atmospheric CO and, collectively, they account for the net loss of ~250 teragrams of CO from the atmosphere each year. However, the physiological role, genetic basis, and ecological distribution of this process remain incompletely resolved. In this work, we addressed these knowledge gaps through culture-based and culture-independent work. We confirmed through shotgun proteomic and transcriptional analysis that the genetically tractable aerobic soil actinobacterium Mycobacterium smegmatis upregulates expression of a form I molydenum-copper carbon monoxide dehydrogenase by 50-fold when exhausted for organic carbon substrates. Whole-cell biochemical assays in wild-type and mutant backgrounds confirmed that this organism aerobically respires CO, including at sub-atmospheric concentrations, using the enzyme. Contrary to current paradigms on CO oxidation, the enzyme did not support chemolithoautotrophic growth and was dispensable for CO detoxification. However, it significantly enhanced long-term survival, suggesting that atmospheric CO serves a supplemental energy source during organic carbon starvation. Phylogenetic analysis indicated that atmospheric CO oxidation is widespread and an ancestral trait of CO dehydrogenases. Homologous enzymes are encoded by 685 sequenced species of bacteria and archaea, including from seven dominant soil phyla, and we confirmed genes encoding this enzyme are abundant and expressed in terrestrial and marine environments. On this basis, we propose a new survival-centric model for the evolution of aerobic CO oxidation and conclude that, like atmospheric H, atmospheric CO is a major energy source supporting persistence of aerobic heterotrophic bacteria in deprived or changeable environments.
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http://dx.doi.org/10.1038/s41396-019-0479-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794299PMC
November 2019

Molecular Basis of Unexpected Specificity of ABC Transporter-Associated Substrate-Binding Protein DppA from Helicobacter pylori.

J Bacteriol 2019 10 20;201(20). Epub 2019 Sep 20.

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

The gastric pathogen has limited ability to use carbohydrates as a carbon source, relying instead on exogenous amino acids and peptides. Uptake of certain peptides by requires an ATP binding cassette (ABC) transporter annotated dipeptide permease (Dpp). The transporter specificity is determined by its cognate substrate-binding protein DppA, which captures ligands in the periplasm and delivers them to the permease. Here, we show that, unlike previously characterized DppA proteins, DppA binds, with micromolar affinity, peptides of diverse amino acid sequences ranging between two and eight residues in length. We present analysis of the 1.45-Å-resolution crystal structure of its complex with the tetrapeptide STSA, which provides a structural rationale for the observed broad specificity. Analysis of the molecular surface revealed a ligand-binding pocket that is large enough to accommodate peptides of up to nine residues in length. The structure suggests that DppA is able to recognize a wide range of peptide sequences by forming interactions primarily with the peptide main chain atoms. The loop that terminates the peptide-binding pocket in DppAs from other bacteria is significantly shorter in the protein, providing an explanation for its ability to bind longer peptides. The subsites accommodating the two N-terminal residues of the peptide ligand make the greatest contribution to the protein-ligand binding energy, in agreement with the observation that dipeptides bind with affinity close to that of longer peptides. The World Health Organization listed as a high-priority pathogen for antibiotic development. The potential of using peptide transporters in drug design is well recognized. We discovered that the substrate-binding protein of the ABC transporter for peptides, termed dipeptide permease, is an unusual member of its family in that it directly binds peptides of diverse amino acid sequences, ranging between two and eight residues in length. We also provided a structural rationale for the observed broad specificity. Since the ability to import peptides as a source of carbon is critical for , our findings will inform drug design strategies based on inhibition or fusion of membrane-impermeant antimicrobials with peptides.
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http://dx.doi.org/10.1128/JB.00400-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755728PMC
October 2019

Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics.

Nat Commun 2019 06 13;10(1):2613. Epub 2019 Jun 13.

Department of Biochemistry and Molecular Biology, The Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.
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http://dx.doi.org/10.1038/s41467-019-10384-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565677PMC
June 2019

Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.

Cell Rep 2019 04;27(5):1528-1540.e7

Department of Physiology, The University of Melbourne, Melbourne, VIC 3010, Australia; Department of Physiology, Monash University, Clayton, VIC 3800, Australia; Metabolism, Diabetes and Obesity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Adipocyte progenitor cells (APCs) provide the reservoir of regenerative cells to produce new adipocytes, although their identity in humans remains elusive. Using FACS analysis, gene expression profiling, and metabolic and proteomic analyses, we identified three APC subtypes in human white adipose tissues. The APC subtypes are molecularly distinct but possess similar proliferative and adipogenic capacities. Adipocytes derived from APCs with high CD34 expression exhibit exceedingly high rates of lipid flux compared with APCs with low or no CD34 expression, while adipocytes produced from CD34 APCs display beige-like adipocyte properties and a unique endocrine profile. APCs were more abundant in gluteofemoral compared with abdominal subcutaneous and omental adipose tissues, and the distribution of APC subtypes varies between depots and in patients with type 2 diabetes. These findings provide a mechanistic explanation for the heterogeneity of human white adipose tissue and a potential basis for dysregulated adipocyte function in type 2 diabetes.
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http://dx.doi.org/10.1016/j.celrep.2019.04.010DOI Listing
April 2019

RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2.

Nat Struct Mol Biol 2019 03 4;26(3):237-247. Epub 2019 Mar 4.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identity during development in multicellular organisms by marking repressed genes and chromatin domains. In addition to four core subunits, PRC2 comprises multiple accessory subunits that vary in their composition during cellular differentiation and define two major holo-PRC2 complexes: PRC2.1 and PRC2.2. PRC2 binds to RNA, which inhibits its enzymatic activity, but the mechanism of RNA-mediated inhibition of holo-PRC2 is poorly understood. Here we present in vivo and in vitro protein-RNA interaction maps and identify an RNA-binding patch within the allosteric regulatory site of human and mouse PRC2, adjacent to the methyltransferase center. RNA-mediated inhibition of holo-PRC2 is relieved by allosteric activation of PRC2 by H3K27me3 and JARID2-K116me3 peptides. Both holo-PRC2.1 and holo-PRC2.2 bind RNA, providing a unified model to explain how RNA and allosteric stimuli antagonistically regulate the enzymatic activity of PRC2.
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http://dx.doi.org/10.1038/s41594-019-0197-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736635PMC
March 2019