scholarly journals Ubiquitin and Not Only Unfolded Domains Drives Toscana Virus Non-Structural NSs Protein Degradation

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1153
Author(s):  
Gianni Gori Savellini ◽  
Luca Bini ◽  
Assunta Gagliardi ◽  
Gabriele Anichini ◽  
Claudia Gandolfo ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Cellular Level ◽  
Structural Protein ◽  
Toscana Virus ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Inhibitory Function ◽  
Ubiquitin System ◽  
Lysine Residues ◽  
The Stability

The non-structural protein NSs of the Phenuiviridae family members appears to have a role in the host immunity escape. The stability of Toscana virus (TOSV) NSs protein was tested by a cycloheximide (CHX) chase approach on cells transfected with NSs deleted versions fused to a reporter gene. The presence of intrinsically disordered regions (IDRs) both at the C- and N-terminus appeared to affect the protein stability. Indeed, the NSsΔC and NSsΔN proteins were more stable than the wild-type NSs counterpart. Since TOSV NSs exerts its inhibitory function by triggering RIG-I for proteasomal degradation, the interaction of the ubiquitin system and TOSV NSs was further examined. Chase experiments with CHX and the proteasome inhibitor MG-132 demonstrated the involvement of the ubiquitin-proteasome system in controlling NSs protein amount expressed in the cells. The analysis of TOSV NSs by mass spectrometry allowed the direct identification of K104, K109, K154, K180, K244, K294, and K298 residues targeted for ubiquitination. Analysis of NSs K-mutants confirmed the presence and the important role of lysine residues located in the central and the C-terminal parts of the protein in controlling the NSs cellular level. Therefore, we directly demonstrated a new cellular pathway involved in controlling TOSV NSs fate and activity, and this opens the way to new investigations among more pathogenic viruses of the Phenuiviridae family.

scholarly journals Variable absorption of mutational trends by prion-forming domains during Saccharomycetes evolution

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9669
Author(s):  
Paul M. Harrison
Keyword(s):  
Large Population ◽  
Selective Constraint ◽  
Alternative States ◽  
Functional Protein ◽  
Selection Pressures ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Intrinsic Protein Disorder ◽  
Lysine Residues

Prions are self-propagating alternative states of protein domains. They are linked to both diseases and functional protein roles in eukaryotes. Prion-forming domains in Saccharomyces cerevisiae are typically domains with high intrinsic protein disorder (i.e., that remain unfolded in the cell during at least some part of their functioning), that are converted to self-replicating amyloid forms. S. cerevisiae is a member of the fungal class Saccharomycetes, during the evolution of which a large population of prion-like domains has appeared. It is still unclear what principles might govern the molecular evolution of prion-forming domains, and intrinsically disordered domains generally. Here, it is discovered that in a set of such prion-forming domains some evolve in the fungal class Saccharomycetes in such a way as to absorb general mutation biases across millions of years, whereas others do not, indicating a spectrum of selection pressures on composition and sequence. Thus, if the bias-absorbing prion formers are conserving a prion-forming capability, then this capability is not interfered with by the absorption of bias changes over the duration of evolutionary epochs. Evidence is discovered for selective constraint against the occurrence of lysine residues (which likely disrupt prion formation) in S. cerevisiae prion-forming domains as they evolve across Saccharomycetes. These results provide a case study of the absorption of mutational trends by compositionally biased domains, and suggest methodology for assessing selection pressures on the composition of intrinsically disordered regions.

scholarly journals Involvement of the Ubiquitin/Proteasome System in Sorting of the Interleukin 2 Receptor β Chain to Late Endocytic Compartments

2001 ◽  
Vol 12 (5) ◽  
pp. 1293-1301 ◽  
Author(s):  
Anna Rocca ◽  
Christophe Lamaze ◽  
Agathe Subtil ◽  
Alice Dautry-Varsat
Keyword(s):  
Degradation Rate ◽  
Interleukin 2 ◽  
Ubiquitin Proteasome System ◽  
Membrane Receptors ◽  
Down Regulation ◽  
Interleukin 2 Receptor ◽  
Ubiquitin System ◽  
Lysine Residues ◽  
Ubiquitin Proteasome ◽  
Β Chain

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor β chain (IL2Rβ) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rβ was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rβ, with little effect on its internalization. In addition, we show that a fraction of IL2Rβ can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rβ targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rβ were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rβ toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

scholarly journals Protein Engineering of Multi-Modular Transcription Factor Alcohol Dehydrogenase Repressor 1 (Adr1p), a Tool for Dissecting In Vitro Transcription Activation

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 497
Author(s):  
Buttinelli ◽  
Panetta ◽  
Bucci ◽  
Frascaria ◽  
Morea ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Transcription Activation ◽  
In Vitro Transcription ◽  
Metabolic Switch ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Transcription Machinery ◽  
Promising Tool ◽  
The Stability

Studying transcription machinery assembly in vitro is challenging because of long intrinsically disordered regions present within the multi-modular transcription factors. One example is alcohol dehydrogenase repressor 1 (Adr1p) from fermenting yeast, responsible for the metabolic switch from glucose to ethanol. The role of each individual transcription activation domain (TAD) has been previously studied, but their interplay and their roles in enhancing the stability of the protein is not known. In this work, we designed five unique miniAdr1 constructs containing either TADs I-II-III or TAD I and III, connected by linkers of different sizes and compositions. We demonstrated that miniAdr1-BL, containing only PAR-TAD I+III with a basic linker (BL), binds the cognate DNA sequence, located in the promoter of the ADH2 (alcohol dehydrogenase 2) gene, and is necessary to stabilize the heterologous expression. In fact, we found that the sequence of the linker between TAD I and III affected the solubility of free miniAdr1 proteins, as well as the stability of their complexes with DNA. miniAdr1-BL is the stable unit able to recognize ADH2 in vitro, and hence it is a promising tool for future studies on nucleosomal DNA binding and transcription machinery assembly in vitro.

scholarly journals Concatenation of 14-3-3 with partner phosphoproteins as a tool to study their interaction

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kristina V. Tugaeva ◽  
Daria I. Kalacheva ◽  
Richard B. Cooley ◽  
Sergei V. Strelkov ◽  
Nikolai N. Sluchanko
Keyword(s):  
Small Molecules ◽  
Limited Proteolysis ◽  
Scanning Calorimetry ◽  
Saxs Data ◽  
X Ray ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
X Ray Scattering ◽  
The Stability ◽  
Partner Proteins

Abstract Regulatory 14-3-3 proteins interact with a plethora of phosphorylated partner proteins, however 14-3-3 complexes feature intrinsically disordered regions and often a transient type of interactions making structural studies difficult. Here we engineer and examine a chimera of human 14-3-3 tethered to a nearly complete partner HSPB6 which is phosphorylated by protein kinase A (PKA). HSPB6 includes a long disordered N-terminal domain (NTD), a phosphorylation motif around Ser16, and a core α-crystallin domain (ACD) responsible for dimerisation. The chosen design enables an unstrained binding of pSer16 in each 1433 subunit and secures the correct 2:2 stoichiometry. Differential scanning calorimetry, limited proteolysis and small-angle X-ray scattering (SAXS) support the proper folding of both the 14-3-3 and ACD dimers within the chimera, and indicate that the chimera retains the overall architecture of the native complex of 14-3-3 and phosphorylated HSPB6 that has recently been resolved using crystallography. At the same time, the SAXS data highlight the weakness of the secondary interface between the ACD dimer and the C-terminal lobe of 14-3-3 observed in the crystal structure. Applied to other 14-3-3 complexes, the chimeric approach may help probe the stability and specificity of secondary interfaces for targeting them with small molecules in the future.

scholarly journals Predicting substitutions to modulate disorder and stability in coiled-coils

2020 ◽  
Author(s):  
Yasaman Karami ◽  
Paul Saighi ◽  
Rémy Vanderhaegen ◽  
Denis Gerlier ◽  
Sonia Longhi ◽  
...  
Keyword(s):  
Protein Function ◽  
Measles Virus ◽  
Rational Design ◽  
Tertiary Structure ◽  
Coiled Coils ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
The Stability ◽  
The Impact

AbstractCoiled-coils are described as stable structural motifs, where two or more helices wind around each other. However, coiled-coils are associated with local mobility and intrinsic disorder. Intrinsically disordered regions (IDRs) in proteins are characterized by lack of stable secondary and tertiary structure under physiological conditions in vitro. They are increasingly recognized as important for protein function. However, characterizing their behaviour in solution and determining precisely the extent of disorder of a protein region remains challenging, both experimentally and computationally. In this work, we propose a computational framework to quantify the extent of disorder within a coiled-coil in solution and to help design substitutions modulating such disorder. Our method relies on the analysis of conformational ensembles generated by relatively short all-atom Molecular Dynamics (MD) simulations. We apply it to the phosphoprotein multimerisation domains (PMD) of Measles virus (MeV) and Nipah virus (NiV), both forming tetrameric left-handed coiled-coils. We show that our method can help quantify the extent of disorder of the C-terminus region of MeV and NiV PMDs, without requiring the input MD trajectory to actually sample the unfolded states of these regions. Moreover, this study provided a conceptual framework for the rational design of substitutions aimed at modulating the stability of the coiled-coils. By assessing the impact of four substitutions known to destabilize coiled-coils, we derive a set of rules to control MeV PMD structural stability and cohesiveness. We therefore design two contrasting substitutions, one increasing the stability of the tetramer and the other increasing its flexibility. Consequently, our method can be considered as a platform to reason about how to design substitutions aimed at regulating flexibility and stability.

scholarly journals Predicting substitutions to modulate disorder and stability in coiled-coils

2020 ◽  
Vol 21 (S19) ◽  
Author(s):  
Yasaman Karami ◽  
Paul Saighi ◽  
Rémy Vanderhaegen ◽  
Denis Gerlier ◽  
Sonia Longhi ◽  
...  
Keyword(s):  
Measles Virus ◽  
Rational Design ◽  
Tertiary Structure ◽  
Md Simulations ◽  
Coiled Coils ◽  
Conformational Space ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
The Stability ◽  
The Impact

Abstract Background Coiled-coils are described as stable structural motifs, where two or more helices wind around each other. However, coiled-coils are associated with local mobility and intrinsic disorder. Intrinsically disordered regions in proteins are characterized by lack of stable secondary and tertiary structure under physiological conditions in vitro. They are increasingly recognized as important for protein function. However, characterizing their behaviour in solution and determining precisely the extent of disorder of a protein region remains challenging, both experimentally and computationally. Results In this work, we propose a computational framework to quantify the extent of disorder within a coiled-coil in solution and to help design substitutions modulating such disorder. Our method relies on the analysis of conformational ensembles generated by relatively short all-atom Molecular Dynamics (MD) simulations. We apply it to the phosphoprotein multimerisation domains (PMD) of Measles virus (MeV) and Nipah virus (NiV), both forming tetrameric left-handed coiled-coils. We show that our method can help quantify the extent of disorder of the C-terminus region of MeV and NiV PMDs from MD simulations of a few tens of nanoseconds, and without requiring an extensive exploration of the conformational space. Moreover, this study provided a conceptual framework for the rational design of substitutions aimed at modulating the stability of the coiled-coils. By assessing the impact of four substitutions known to destabilize coiled-coils, we derive a set of rules to control MeV PMD structural stability and cohesiveness. We therefore design two contrasting substitutions, one increasing the stability of the tetramer and the other increasing its flexibility. Conclusions Our method can be considered as a platform to reason about how to design substitutions aimed at regulating flexibility and stability.

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