scholarly journals Transient exposure of a buried phosphorylation site in an autoinhibited protein

2021 ◽  
Author(s):  
Simone Orioli ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Protein Function ◽  
Molten Globule ◽  
Phosphorylation Site ◽  
Structural Disorder ◽  
Helical Structures ◽  
Post Translational Modifications ◽  
Cis Acting ◽  
Functional Sites ◽  
Inhibitory Domain ◽  
Regulate Protein

Autoinhibition is a mechanism used to regulate protein function, often by making functional sites inaccessible through the interaction with a cis-acting inhibitory domain. Such autoinhibitory domains often display a substantial degree of structural disorder when unbound, and only become structured in the inhibited state. This structural dynamics makes it difficult to study the structural origin of regulation, including effects of regulatory post-translational modifications. Here, we study the autoinhibition of the Dbl Homology domain in the protein Vav1 by the so-called acidic inhibitory domain. We use molecular simulations to study the process by which a mostly unstructured inhibitory domain folds upon binding and how transient exposure of a key buried tyrosine residue makes it accessible for phosphorylation. We show that the inhibitory domain, which forms a helix in the bound and inhibited stated, samples helical structures already before binding and that binding occurs via a molten-globule-like intermediate state. Together, our results shed light on key interactions that enable the inhibitory domain to sample a finely-tuned equilibrium between an inhibited and a kinase-accessible state.

scholarly journals iProteinDB: an integrative database of Drosophila post-translational modifications

2018 ◽  
Author(s):  
Yanhui Hu ◽  
Richelle Sopko ◽  
Verena Chung ◽  
Romain A. Studer ◽  
Sean D. Landry ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Large Scale ◽  
Model Organisms ◽  
General Strategy ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Sites ◽  
Evolutionarily Conserved ◽  
And Function

AbstractPost-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing stability, protein interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, commonly serine, threonine and tyrosine. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that many phosphorylation sites may be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites with regards to regulation and function. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus laevis, Danio rerio, and Caenorhabditis elegans.

scholarly journals Ubiquitylation of ABA Receptors and Protein Phosphatase 2C Coreceptors to Modulate ABA Signaling and Stress Response

2021 ◽  
Vol 22 (13) ◽  
pp. 7103
Author(s):  
Alberto Coego ◽  
Jose Julian ◽  
Jorge Lozano-Juste ◽  
Gaston A. Pizzio ◽  
Abdulwahed F. Alrefaei ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Aba Signaling ◽  
Post Translational Modifications ◽  
Core Components ◽  
Regulate Protein ◽  
Branched Chains ◽  
Aba Receptors

Post-translational modifications play a fundamental role in regulating protein function and stability. In particular, protein ubiquitylation is a multifaceted modification involved in numerous aspects of plant biology. Landmark studies connected the ATP-dependent ubiquitylation of substrates to their degradation by the 26S proteasome; however, nonproteolytic functions of the ubiquitin (Ub) code are also crucial to regulate protein interactions, activity, and localization. Regarding proteolytic functions of Ub, Lys-48-linked branched chains are the most common chain type for proteasomal degradation, whereas promotion of endocytosis and vacuolar degradation is triggered through monoubiquitylation or Lys63-linked chains introduced in integral or peripheral plasma membrane proteins. Hormone signaling relies on regulated protein turnover, and specifically the half-life of ABA signaling components is regulated both through the ubiquitin-26S proteasome system and the endocytic/vacuolar degradation pathway. E3 Ub ligases have been reported that target different ABA signaling core components, i.e., ABA receptors, PP2Cs, SnRK2s, and ABFs/ABI5 transcription factors. In this review, we focused specifically on the ubiquitylation of ABA receptors and PP2C coreceptors, as well as other post-translational modifications of ABA receptors (nitration and phosphorylation) that result in their ubiquitination and degradation.

scholarly journals Protein abundance is key to distinguish promiscuous from functional phosphorylation based on evolutionary information

2012 ◽  
Vol 367 (1602) ◽  
pp. 2594-2606 ◽  
Author(s):  
Emmanuel D. Levy ◽  
Stephen W. Michnick ◽  
Christian R. Landry
Keyword(s):  
Protein Function ◽  
Evolutionary Conservation ◽  
Evolutionary Information ◽  
Key Factors ◽  
Evolutionary Patterns ◽  
Functional Sites ◽  
Saccharomyces Bayanus ◽  
Regulate Protein ◽  
Protein Conservation ◽  
The Impact

In eukaryotic cells, protein phosphorylation is an important and widespread mechanism used to regulate protein function. Yet, of the thousands of phosphosites identified to date, only a few hundred at best have a characterized function. It was recently shown that these functional sites are significantly more conserved than phosphosites of unknown function, stressing the importance of considering evolutionary conservation in assessing the global functional landscape of phosphosites. This leads us to review studies that examined the impact of phosphorylation on evolutionary conservation. While all these studies have shown that conservation is greater among phosphorylated sites compared with non-phosphorylated ones, the magnitude of this difference varies greatly. Further, not all studies have considered key factors that may influence the rate of phosphosite evolution. Such key factors are their localization in ordered or disordered regions, their stoichiometry or the abundance of their corresponding protein. Here we take into account all of these factors simultaneously, which reveals remarkable evolutionary patterns. First, while it is well established that protein conservation increases with abundance, we show that phosphosites partly follow an opposite trend. More precisely, Saccharomyces cerevisiae phosphosites present among abundant proteins are 1.5 times more likely to diverge in the closely related species Saccharomyces bayanus when compared with phosphosites present in the 5 per cent least abundant proteins. Second, we show that conservation is coupled to stoichiometry, whereby sites frequently phosphorylated are more conserved than those rarely phosphorylated. Finally, we provide a model of functional and noisy or ‘accidental’ phosphorylation that explains these observations.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

scholarly journals Post translational modifications of milk proteins in geographically diverse goat breeds

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. K. Rout ◽  
M. Verma
Keyword(s):  
Protein Function ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Goat Milk ◽  
Peptide Sequence ◽  
Cow Milk ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Dpp Iv

AbstractGoat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.

scholarly journals In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

1997 ◽  
Vol 139 (5) ◽  
pp. 1197-1207 ◽  
Author(s):  
N. Doane Chilcoat ◽  
Aaron P. Turkewitz
Keyword(s):  
Membrane Fusion ◽  
Protein Function ◽  
Potential Role ◽  
Secretory Granules ◽  
Rabbit Muscle ◽  
Paramecium Tetraurelia ◽  
Post Translational Modifications ◽  
In Vivo Analysis ◽  
And Function

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

scholarly journals Functional Importance of Hydrophobic Patches on the Ebola Virus VP35 IFN-Inhibitory Domain

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2316
Author(s):  
Nodoka Kasajima ◽  
Keita Matsuno ◽  
Hiroko Miyamoto ◽  
Masahiro Kajihara ◽  
Manabu Igarashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ebola Virus ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Genome Replication ◽  
Type I ◽  
Multifunctional Protein ◽  
Functional Sites ◽  
Inhibitory Function ◽  
Inhibitory Domain

Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-β promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.

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