Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Mario Oroshi
LAB HEAD: Matthias Mann
PROVIDER: PXD021286 | Pride | 2020-09-09
REPOSITORIES: Pride
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Project description:Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
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2024-07-13 | E-MTAB-14214 | biostudies-arrayexpress
Project description:Condensation (C) domains are the key component linking different monomers together, typically forming peptide bonds and occasionally ester bonds, during the nonribosomal peptide synthetase (NRPS). While A domains have been well characterised due to their role in selectivity of the monomers and functioning as a gate keeper in the NRPS biosynthesis, C domains have been a subject of debate as they have not demonstrated signs of “A-domain like” side chain selectivity of its acceptor side. Here, we report our biochemical and structural characterisation of the selectivity of the fuscachelin C3-domain showing that it is not broadly flexible for monomers at the acceptor site, suggesting the need to consider C-domain mutation regarding future NRPS engineering.
2023-06-02 | PXD040334 | Pride
Project description:Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
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Project description:Mass spectrometry-based proteomics has become an integral approach for characterising ubiquitin chain-linkage compositions and architectures. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-minute LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages (BMDMs) and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical ubiquitin chain types (K29 and K33) in heart and muscle. Ubiquitin chain topology profiling using tandem ubiquitin binding entities, directed at K29/K33 ubiquitin chains (TRABID), and mass spectrometry analysis identified several mitochondrial proteins as putatively associated with these atypical ubiquitin chain types. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues. Our results contribute to our understanding of in vivo ubiquitin chain-linkage composition in murine tissues.
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Project description:Identification of vitamin K side chain cleavage enzyme
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Project description:Bacterial protein glycosylation can be mediated by oligosaccharyltransferases (OTases) that transfer a preassembled lipid-linked oligosaccharide or polysaccharide en bloc to acceptor proteins. O-linking OTases transfer O-antigen or capsular polysaccharides to the side chains of serine or threonine residues. Three major families of bacterial O-linking OTases have been described so far: PglL, PglS, and TfpO. TfpO enzymes are limited to transferring only short glycans whereas there are no clear upper limits for the other two families. Herein, we describe the discovery of a novel family of bacterial O-linking OTases from Moraxellacea bacteria that are similar in size and sequence to TfpO enzymes but can transfer long-chain bacterial glycans to acceptor proteins. Bioinformatic analyses show that these enzymes cluster in different clades than known bacterial OTases. Using a representative enzyme from Moraxella osloensis termed TfpMMo, we determine that the enzyme glycosylates the C-terminal amino acid side chain of a pilin protein and find that pilin fragments as short as three amino acids are substrates for the OTase. The ability of TfpMMo to transfer long-chain polysaccharide shows that this ability is not limited to the PglS and PglL families. TfpMMo is also shown to have broad substrate specificity and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. The glycan substrate promiscuity of TfpMMo could allow this enzyme to be used to produce bacterial glycoconjugate vaccines. The discovery of a new class of O-linking OTase furthers our understanding of the mechanisms that underly glycan specificity by these and other O-linking OTases and enables more comparative studies of this important enzyme family.
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Project description:Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C-22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyses the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one (SDO) to a conjugated 22,(E)24-diene. We suggest an 44 architecture of the 440 kDa enzyme, with each subunit covalently binding an FMN cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by SDO but cannot be oxidised even with strong oxidising agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.
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Project description:Proctor2007 - Age related decline of proteolysis, ubiquitin-proteome system
This
is a stochastic model of the ubiquitin-proteasome system for a
generic pool of native proteins (NatP), which have a half-life of
about 10 hours under normal conditions. It is assumed that these
proteins are only degraded after they have lost their native
structure due to a damage event. This is represented in the model
by the misfolding reaction which depends on the level of reactive
oxygen species (ROS) in the cell. Misfolded proteins (MisP) are
first bound by an E3 ubiquitin ligase. Ubiquitin (Ub) is
activated by E1 (ubiquitin-activating enzyme) and then passed to
E2 (ubiquitin-conjugating enzyme). The E2 enzyme then passes the
ubiquitin molecule to the E3/MisP complex with the net effect
that the misfolded protein is monoubiquitinated and both E2 and
E3 are released. Further ubiquitin molecules are added in a
step-wise manner. When the chain of ubiquitin molecules is of
length 4 or more, the polyubiquitinated misfolded protein may
bind to the proteasome. The model also includes de-ubiquitinating
enzymes (DUB) which cleave ubiquitin molecules from the chain in
a step-wise manner. They work on chains attached to misfolded
proteins both unbound and bound to the proteasomes. Misfolded
proteins bound to the proteasome may be degraded releasing
ubiquitin. Misfolded proteins including ubiquitinated proteins
may also aggregate. Aggregates (AggP) may be sequestered
(Seq_AggP) which takes them out of harm's way or they may bind to
the proteasome (AggP_Proteasome). Proteasomes bound by aggregates
are no longer available for protein degradation.
Figure
2 and Figure 3 has been simulated using Gillespie2.
This model is described in the article:
An in silico model of the
ubiquitin-proteasome system that incorporates normal
homeostasis and age-related decline.
Proctor CJ, Tsirigotis M, Gray
DA.
BMC Syst Biol 2007; 1: 17
Abstract:
BACKGROUND: The ubiquitin-proteasome system is responsible
for homeostatic degradation of intact protein substrates as
well as the elimination of damaged or misfolded proteins that
might otherwise aggregate. During ageing there is a decline in
proteasome activity and an increase in aggregated proteins.
Many neurodegenerative diseases are characterised by the
presence of distinctive ubiquitin-positive inclusion bodies in
affected regions of the brain. These inclusions consist of
insoluble, unfolded, ubiquitinated polypeptides that fail to be
targeted and degraded by the proteasome. We are using a systems
biology approach to try and determine the primary event in the
decline in proteolytic capacity with age and whether there is
in fact a vicious cycle of inhibition, with accumulating
aggregates further inhibiting proteolysis, prompting
accumulation of aggregates and so on. A stochastic model of the
ubiquitin-proteasome system has been developed using the
Systems Biology Mark-up Language (SBML). Simulations are
carried out on the BASIS (Biology of Ageing e-Science
Integration and Simulation) system and the model output is
compared to experimental data wherein levels of ubiquitin and
ubiquitinated substrates are monitored in cultured cells under
various conditions. The model can be used to predict the
effects of different experimental procedures such as inhibition
of the proteasome or shutting down the enzyme cascade
responsible for ubiquitin conjugation. RESULTS: The model
output shows good agreement with experimental data under a
number of different conditions. However, our model predicts
that monomeric ubiquitin pools are always depleted under
conditions of proteasome inhibition, whereas experimental data
show that monomeric pools were depleted in IMR-90 cells but not
in ts20 cells, suggesting that cell lines vary in their ability
to replenish ubiquitin pools and there is the need to
incorporate ubiquitin turnover into the model. Sensitivity
analysis of the model revealed which parameters have an
important effect on protein turnover and aggregation kinetics.
CONCLUSION: We have developed a model of the
ubiquitin-proteasome system using an iterative approach of
model building and validation against experimental data. Using
SBML to encode the model ensures that it can be easily modified
and extended as more data become available. Important aspects
to be included in subsequent models are details of ubiquitin
turnover, models of autophagy, the inclusion of a pool of
short-lived proteins and further details of the aggregation
process.
This model is hosted on
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BIOMD0000000105.
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2024-09-02 | BIOMD0000000105 | BioModels