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1.
Elife ; 132024 Jul 10.
Article in English | MEDLINE | ID: mdl-38984715

ABSTRACT

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

2.
J Biol Chem ; 299(8): 104948, 2023 08.
Article in English | MEDLINE | ID: mdl-37354974

ABSTRACT

Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to a 28-subunit hollow core particle (CP) where proteolysis occurs. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 (proteasome 26S subunit, non-ATPase 2) for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a tobacco etch virus protease cut site at the C terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with tobacco etch virus protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry.


Subject(s)
Cytological Techniques , Proteasome Endopeptidase Complex , Animals , Cattle , Humans , Cell Line , Cytoplasm/metabolism , Mammals/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitin/metabolism , Cytological Techniques/methods
3.
Biochem J ; 477(12): 2193-2219, 2020 06 26.
Article in English | MEDLINE | ID: mdl-32478812

ABSTRACT

The Baculoviridae family of viruses encode a viral Ubiquitin (vUb) gene. Though the vUb is homologous to the host eukaryotic Ubiquitin (Ub), its preservation in the viral genome indicates unique functions that are not compensated by the host Ub. We report the structural, biophysical, and biochemical properties of the vUb from Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV). The packing of central helix α1 to the beta-sheet ß1-ß5 is different between vUb and Ub. Consequently, its stability is lower compared with Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin-binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54), and the deubiquitinating enzymes are ineffective against the K54-linked polyubiquitin chains. We propose that the modification of host/viral proteins with the K54-linked chains is an effective way selected by the virus to protect the vUb signal from host DeUbiquitinases.


Subject(s)
Closterovirus/metabolism , Deubiquitinating Enzymes/metabolism , Polyubiquitin/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/metabolism , Ubiquitination , Viral Proteins/metabolism , Amino Acid Sequence , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/genetics , HEK293 Cells , Humans , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Polyubiquitin/chemistry , Protein Conformation , Sequence Homology , Viral Proteins/chemistry
4.
J Mol Biol ; 432(7): 1952-1977, 2020 03 27.
Article in English | MEDLINE | ID: mdl-32001251

ABSTRACT

When the herpes simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called Promyelocytic leukemia protein nuclear bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) infected cell polypeptide 0 (ICP0) that degrades PML NBs to alleviate the repression. The molecular details of the mechanism used by ICP0 to target PML NBs are unclear. Here, we identify a bona fide SUMO-interacting motif in ICP0 (SIM-like sequence [SLS] 4) that is essential and sufficient to target SUMOylated proteins in PML NBs such as the PML and Sp100. We shown that phosphorylation of SLS4 creates new salt bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity, and switches ICP0 into a potent STUbL. HSV activates the Ataxia-telangiectasia-mutated kinase-Checkpoint kinase 2 (ATM-Chk2) pathway to regulate the cell cycle of the host. We report that the activated Chk2 also phosphorylates ICP0 at SLS4 and enhances its STUbL activity. Our results uncover that a viral STUbL counters antiviral response by exploiting an unprecedented cross-talk of three post-translational modifications: ubiquitination, SUMOylation, and phosphorylation.


Subject(s)
Checkpoint Kinase 2/metabolism , Herpes Simplex/virology , Herpesvirus 1, Human/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Viral Proteins/metabolism , Checkpoint Kinase 2/chemistry , Checkpoint Kinase 2/genetics , HEK293 Cells , Humans , Phosphorylation , Protein Conformation , Protein Domains , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/genetics , Sumoylation , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication
5.
Nat Commun ; 10(1): 452, 2019 01 28.
Article in English | MEDLINE | ID: mdl-30692525

ABSTRACT

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue 'cystatin motif' (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two ß-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse ß-hairpin proteins.


Subject(s)
Amino Acid Motifs/genetics , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Amino Acid Sequence , Cystatin B/chemistry , Cystatin B/genetics , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutation , Protein Engineering/methods , Sequence Homology, Amino Acid
6.
PLoS One ; 9(3): e89079, 2014.
Article in English | MEDLINE | ID: mdl-24594932

ABSTRACT

Cellular and molecular approaches are being explored to find a biomarker which can predict the development of radiation induced acute toxicity prior to radiation therapy. SNPs in radiation responsive genes may be considered as an approach to develop tools for finding the inherited basis of clinical radiosensitivity. The current study attempts to screen single nucleotide polymorphisms/deletions in DNA damage response, DNA repair, profibrotic cytokine as well as antioxidant response genes and its predictive potential with the normal tissue adverse reactions from 183 head and neck cancer patients undergoing platinum based chemoradiotherapy or radiotherapy alone. We analysed 22 polymorphisms in 17 genes having functional relevance to radiation response. Radiation therapy induced oral mucositis and skin erythema was considered as end point for clinical radiosensitivity. Direct correlation of heterozygous and mutant alleles with acute reactions as well as haplotype correlation revealed NBN variants to be of predictive significance in analysing oral mucositis prior to radiotherapy. In addition, genetic linkage disequilibrium existed in XRCC1 polymorphisms for >grade 2 oral mucositis and skin reaction indicating the complex inheritance pattern. The current study indicates an association for polymorphism in NBN with normal tissue radiosensitivity and further warrants the replication of such studies in a large set of samples.


Subject(s)
Chemoradiotherapy/adverse effects , Genetic Association Studies , Head and Neck Neoplasms/genetics , Polymorphism, Genetic , Radiation Tolerance/genetics , Adult , Aged , Aged, 80 and over , Alleles , Confounding Factors, Epidemiologic , Demography , Female , Genetic Predisposition to Disease , Haplotypes/genetics , Humans , Linkage Disequilibrium/genetics , Male , Middle Aged , Multivariate Analysis , Polymorphism, Single Nucleotide/genetics , Risk Factors
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