Suppression of RNA Interference by Adenovirus Virus-Associated RNA

ABSTRACT We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3′ strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.

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Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

10.1101/2019.12.16.878546 ◽

2019 ◽

Author(s):

Cassandra K. Hayne ◽

Casey A. Schmidt ◽

A. Gregory Matera ◽

Robin E. Stanley

Keyword(s):

Animal Cells ◽

Trna Splicing ◽

Polynucleotide Kinase ◽

Genes Encoding ◽

Intron Removal ◽

And Function ◽

Insight Into ◽

Negative Modulator

ABSTRACTThe splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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Mechanistic insight into DsyB/DSYB, key enzymes in marine dimethylsulfoniopropionate synthesis

10.21203/rs.3.rs-454893/v1 ◽

2021 ◽

Author(s):

Jonathan Todd ◽

Chun-Yang Li ◽

Jason Crack ◽

Simone Newton-Payne ◽

Andrew Murphy ◽

...

Keyword(s):

Marine Algae ◽

Nucleophilic Attack ◽

Mechanistic Insight ◽

Signalling Molecule ◽

Major Nutrient ◽

Methyltransferase Gene ◽

Key Enzyme ◽

Algae And Bacteria ◽

Insight Into

Abstract Marine algae and bacteria produce eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth’s surface oceans every year. DMSP is an anti-stress compound and, once released into the environment, a major nutrient, signalling molecule and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria. The S-directed S-adenosylmethionine-dependent methyltransferase (SAM-MT) 4-methylthio-2-hydroxybutyrate (MTHB) S-methyltransferase, encoded by the dsyB/DSYB gene, is the key enzyme of this pathway, generating S-adenosylhomocysteine (SAH) and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB). dsyB/DSYB, present in most DMSP-producing bacteria and haptophyte and dinoflagellate algae with the highest known DMSP concentrations, is shown to be far more abundant and transcribed in marine environments than any other known DMSP synthesis pathway S-methyltransferase gene. Furthermore, we demonstrate in vitro activity of the bacterial DsyB enzyme from Nisaea denitrificans, and provide its crystal structure in complex with SAM and SAH-MTHB, which together provide the first mechanistic insights into a DMSP synthesis enzyme. Structural and mutational analyses imply that DsyB adopts a novel mechanism, distinct from any previously reported SAM-MT, in which the DsyB residue Tyr142 activates the sulfur atom of MTHB for nucleophilic attack on the SAM methyl group. Sequence analysis suggests that this mechanism is common to all bacterial DsyB enzymes and also, importantly, eukaryotic DSYB enzymes from e.g., algae that are the major DMSP producers in Earth’s surface oceans.

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Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

Nucleic Acids Research ◽

10.1093/nar/gkaa438 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7609-7622 ◽

Cited By ~ 3

Author(s):

Cassandra K Hayne ◽

Casey A Schmidt ◽

Maira I Haque ◽

A Gregory Matera ◽

Robin E Stanley

Keyword(s):

Animal Cells ◽

Trna Splicing ◽

Polynucleotide Kinase ◽

Genes Encoding ◽

Intron Removal ◽

And Function ◽

Insight Into ◽

Negative Modulator

Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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Filamentation of the bacterial bi-functional alcohol/aldehyde dehydrogenase AdhE is essential for substrate channeling and enzymatic regulation

10.1101/2019.12.19.882415 ◽

2019 ◽

Author(s):

Pauline Pony ◽

Chiara Rapisarda ◽

Laurent Terradot ◽

Esther Marza ◽

Rémi Fronzes

Keyword(s):

Metabolic Enzyme ◽

Substrate Channeling ◽

Bacterial Physiology ◽

Macromolecular Assemblies ◽

Enzymatic Regulation ◽

Oligomeric Form ◽

Key Enzyme ◽

And Function

AbstractAcetaldehyde – alcohol dehydrogenase (AdhE) enzymes are a key metabolic enzyme in bacterial physiology and pathogenicity. They convert acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in anaerobic environment. This two-step reaction is associated to NAD+ regeneration, essential for glycolysis. The bifunctional AdhE enzyme is conserved in all bacterial kingdoms but also in more phylogenetically distant microorganisms such as green microalgae. In synthetic biology and biotechnology, because of its central role in bacterial alcoholic fermentation, AdhE raised a lot of attention as a key enzyme to produce ethanol from bacterial cultures.AdhE is commonly found as an oligomeric form called spirosomes. While these helical macromolecular assemblies are conserved, their function remains elusive. We used cryo-electron microscopy to obtain structures of Escherichia coli spirosomes in different conformational states. We confirm that spirosomes contain active AdhE monomers and show that AdhE filamentation is essential for its activity in vitro and function in vivo. The detailed analysis of these structures provides insight showing that AdhE filamentation is essential for substrate channeling within the filament and for the regulation of enzyme activity. These new data will help to design molecules or mutations that control AdhE activity to fight bacterial pathogens or to optimize ethanol production in biotechnology.

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Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins

International Journal of Molecular Sciences ◽

10.3390/ijms19113418 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3418 ◽

Cited By ~ 2

Author(s):

Henrik O’Brien ◽

Shingo Kanemura ◽

Masaki Okumura ◽

Robert Baskin ◽

Pradip Bandyopadhyay ◽

...

Keyword(s):

Protein Disulfide Isomerase ◽

Venom Gland ◽

Cone Snail ◽

Disulfide Isomerase ◽

Folding Rates ◽

Cone Snails ◽

And Function ◽

Protein Disulfide ◽

Insight Into

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

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Quantitative assessment of successive carbohydrate additions to the clustered O-glycosylation sites of IgA1 by glycosyltransferases

Glycobiology ◽

10.1093/glycob/cwaa111 ◽

2020 ◽

Author(s):

Tyler J Stewart ◽

Kazuo Takahashi ◽

Nuo Xu ◽

Amol Prakash ◽

Rhubell Brown ◽

...

Keyword(s):

Molecular Mechanisms ◽

Structure And Function ◽

Reaction Products ◽

Technological Advances ◽

Glycosylation Sites ◽

Relative Quantitation ◽

And Function ◽

Insight Into ◽

Subsequent Activity

Abstract Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling, and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or IgA1. Here we describe a series of nanoLC–MS analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgA1. By utilizing nanoLC–MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgA1 O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a polypeptide GalNAc-transferase and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and α2,3- and α2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgA1, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgA1 O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.

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Identification of the Pseudorabies Virus Promoter Required for Latency-Associated Transcript Gene Expression in the Natural Host

Journal of Virology ◽

10.1128/jvi.74.14.6333-6338.2000 ◽

2000 ◽

Vol 74 (14) ◽

pp. 6333-6338 ◽

Cited By ~ 11

Author(s):

Ling Jin ◽

William M. Schnitzlein ◽

Gail Scherba

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Pseudorabies Virus ◽

Consensus Sequence ◽

Neuronal Cells ◽

Natural Host ◽

Lytic Infection ◽

Latently Infected ◽

And Function

ABSTRACT Expression of the latency-associated transcript (LAT) gene is a hallmark of alphaherpesvirus latency, and yet its control and function remain an enigma. Resolution of this problem will require verification and subsequent elimination or disabling of elements regulating LAT gene transcription so that the influence of the resultant RNA can be evaluated. Toward this end, we generated a novel pseudorabies virus (PrV) recombinant in which a 282-bp region containing the LAP1 (first latency-active promoter) consensus sequence was replaced by a reporter cassette. Despite this substitution, replication of the recombinant was comparable to that of the parental and rescuant viruses both in cultured mammalian cells and in the natural host, swine. Furthermore, production of the LAT gene-associated 2.0- and 8.0-kb RNAs during an in vitro lytic infection of cultured neuronal cells was unaffected. However, the otherwise constitutively produced and processed 8.4-kb LAT was not detected in porcine trigeminal ganglia latently infected with this novel recombinant, although the viral genome was shown to be present. Therefore, LAP1 is apparently the basal promoter for PrV LAT gene expression during viral latency but is not required for such activity during an in vitro lytic infection of neuronal cells. More importantly, the ability of PrV to persist in a latent state in the absence of LAT suggests that other factors are responsible for this event in the natural host.

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Conformational flexibility and allosteric regulation of cathepsin K

Biochemical Journal ◽

10.1042/bj20100337 ◽

2010 ◽

Vol 429 (2) ◽

pp. 379-389 ◽

Cited By ~ 45

Author(s):

Marko Novinec ◽

Lidija Kovačič ◽

Brigita Lenarčič ◽

Antonio Baici

Keyword(s):

Dermatan Sulfate ◽

Molecular Level ◽

Cathepsin K ◽

Conformational Flexibility ◽

Kinetic Properties ◽

Collagenolytic Activity ◽

Key Enzyme ◽

Multiple Conformations ◽

Insight Into

The human cysteine peptidase cathepsin K is a key enzyme in bone homoeostasis and other physiological functions. In the present study we investigate the mechanism of cathepsin K action at physiological plasma pH and its regulation by modifiers that bind outside of the active site. We show that at physiological plasma pH the enzyme fluctuates between multiple conformations that are differently susceptible to macromolecular inhibitors and can be manipulated by varying the ionic strength of the medium. The behaviour of the enzyme in vitro can be described by the presence of two discrete conformations with distinctive kinetic properties and different susceptibility to inhibition by the substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin. We identify and characterize sulfated glycosaminoglycans as natural allosteric modifiers of cathepsin K that exploit the conformational flexibility of the enzyme to regulate its activity and stability against autoproteolysis. All sulfated glycosaminoglycans act as non-essential activators in assays using low-molecular-mass substrates. Chondroitin sulfate and dermatan sulfate bind at one site on the enzyme, whereas heparin binds at an additional site and has a strongly stabilizing effect that is unique among human glycosaminoglycans. All glycosaminoglycans stimulate the elastinolytic activity of cathepsin K at physiological plasma pH, but only heparin also increases the collagenolytic activity of the enzyme under these conditions. Altogether these results provide novel insight into the mechanism of cathepsin K function at the molecular level and its regulation in the extracellular space.

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PIP4Kβ interacts with and modulates nuclear localization of the high-activity PtdIns5P-4-kinase isoform PIP4Kα

Biochemical Journal ◽

10.1042/bj20100341 ◽

2010 ◽

Vol 430 (2) ◽

pp. 223-235 ◽

Cited By ~ 57

Author(s):

Yvette Bultsma ◽

Willem-Jan Keune ◽

Nullin Divecha

Keyword(s):

Crystal Structure ◽

Rna Interference ◽

High Activity ◽

Nuclear Localization ◽

Functional Interaction ◽

Specific Antibodies ◽

Tumour Suppressor P53 ◽

Insight Into

The β-isoform of PIP4K (PtdIns5P-4-kinase) regulates the levels of nuclear PtdIns5P, which in turn modulates the acetylation of the tumour suppressor p53. The crystal structure of PIP4Kβ demonstrated that it can form a homodimer with the two subunits arranged in opposite orientations. Using MS, isoform-specific antibodies against PIP4Ks, RNAi (RNA interference) suppression and overexpression studies, we show that PIP4Kβ interacts in vitro and in vivo with the PIP4Kα isoform. As the two isoforms phosphorylate the same substrate to generate the same product, the interaction could be considered to be functionally redundant. However, contrary to expectation, we find that PIP4Kβ has 2000-fold less activity towards PtdIns5P compared with PIP4Kα, and that the majority of PIP4K activity associated with PIP4Kβ comes from its interaction with PIP4Kα. Furthermore, PIP4Kβ can modulate the nuclear localization of PIP4Kα, and PIP4Kα has a role in regulating PIP4Kβ functions. The results of the present study suggest a rationale for the functional interaction between PIP4Kα and PIP4Kβ and provide insight into how the relative levels of the two enzymes may be important in their physiological and pathological roles.

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Biophysical characterization of polyomavirus minor capsid proteins

Biological Chemistry ◽

10.1515/hsz-2014-0114 ◽

2014 ◽

Vol 395 (7-8) ◽

pp. 871-880 ◽

Cited By ~ 3

Author(s):

Oliver Burkert ◽

Susanne Kreßner ◽

Ludwig Sinn ◽

Sven Giese ◽

Claudia Simon ◽

...

Keyword(s):

Capsid Proteins ◽

Biophysical Characterization ◽

Helical Content ◽

Assembly Unit ◽

Murine Polyomavirus ◽

Capsid Protein Vp1 ◽

And Function ◽

Insight Into

Abstract The murine polyomavirus encodes three structural proteins, VP1, VP2 and VP3, which together form the viral capsid. The outer shell of this capsid is composed of the major capsid protein VP1, the inner shell consists of VP2 and its N-terminally truncated form VP3. These two minor capsid proteins interact with their identical C-terminal part in the central cavity of VP1 pentamers, building the capsid assembly unit. While the VP1 structure and functions are well studied, VP2 and VP3 are poorly understood. In order to get a detailed insight into the structure and function of the minor capsid proteins, they were produced recombinantly in Escherichia coli as inclusion bodies and refolded in vitro. The success of refolding was strictly dependent on the presence of detergent in the refolding buffer. VP2 and VP3 are monomeric and their structures exhibit a high α-helical content. The function of both proteins could be monitored by complex formation with VP1. Furthermore, we could demonstrate a hemolytic activity of VP2/VP3 in vitro, which fits well into a postulated membrane interaction of VP2 during viral infection.

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