scholarly journals Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding

2020 ◽  
Vol 295 (35) ◽  
pp. 12343-12352 ◽  
Author(s):  
Ryo Iwagishi ◽  
Rika Tanaka ◽  
Munenosuke Seto ◽  
Tomoyo Takagi ◽  
Naoko Norioka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Molecular Mechanisms ◽  
Alternative Exon ◽  
Ectodomain Shedding ◽  
Post Translational Modification ◽  
Charged Amino Acids ◽  
Juxtamembrane Region ◽  
Protein Variants ◽  
Variant Protein

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.

scholarly journals To cut or not to cut: New rules for proteolytic shedding of membrane proteins

2020 ◽  
Vol 295 (35) ◽  
pp. 12353-12354
Author(s):  
Stefan F. Lichtenthaler ◽  
Edgar Meinl
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Extracellular Domain ◽  
Ectodomain Shedding ◽  
Juxtamembrane Domain ◽  
Charged Amino Acids ◽  
And Function ◽  
Proteolytic Shedding

Sheddases are specialized proteases that control the abundance and function of membrane proteins by cleaving their substrate's extracellular domain (ectodomain), a process known as shedding. Hundreds of shedding substrates have been identified, but little is known about the mechanisms that govern ectodomain shedding. Iwagishi et al. now report that negatively charged amino acids in the membrane-proximal juxtamembrane domain of substrates make them resistant to shedding by the metalloprotease ADAM17. These findings will help researchers better understand the regulation of shedding and may aid in the development of drugs targeting sheddases.

scholarly journals iDPGK: characterization and identification of lysine phosphoglycerylation sites based on sequence-based features

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Kai-Yao Huang ◽  
Fang-Yu Hung ◽  
Hui-Ju Kao ◽  
Hui-Hsuan Lau ◽  
Shun-Long Weng
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Training Dataset ◽  
Post Translational Modification ◽  
Web Based ◽  
Charged Amino Acids ◽  
Pair Composition ◽  
Weighted Matrix ◽  
Svm Model ◽  
Effective Performance

Abstract Background Protein phosphoglycerylation, the addition of a 1,3-bisphosphoglyceric acid (1,3-BPG) to a lysine residue of a protein and thus to form a 3-phosphoglyceryl-lysine, is a reversible and non-enzymatic post-translational modification (PTM) and plays a regulatory role in glucose metabolism and glycolytic process. As the number of experimentally verified phosphoglycerylated sites has increased significantly, statistical or machine learning methods are imperative for investigating the characteristics of phosphoglycerylation sites. Currently, research into phosphoglycerylation is very limited, and only a few resources are available for the computational identification of phosphoglycerylation sites. Result We present a bioinformatics investigation of phosphoglycerylation sites based on sequence-based features. The TwoSampleLogo analysis reveals that the regions surrounding the phosphoglycerylation sites contain a high relatively of positively charged amino acids, especially in the upstream flanking region. Additionally, the non-polar and aliphatic amino acids are more abundant surrounding phosphoglycerylated lysine following the results of PTM-Logo, which may play a functional role in discriminating between phosphoglycerylation and non-phosphoglycerylation sites. Many types of features were adopted to build the prediction model on the training dataset, including amino acid composition, amino acid pair composition, positional weighted matrix and position-specific scoring matrix. Further, to improve the predictive power, numerous top features ranked by F-score were considered as the final combination for classification, and thus the predictive models were trained using DT, RF and SVM classifiers. Evaluation by five-fold cross-validation showed that the selected features was most effective in discriminating between phosphoglycerylated and non-phosphoglycerylated sites. Conclusion The SVM model trained with the selected sequence-based features performed well, with a sensitivity of 77.5%, a specificity of 73.6%, an accuracy of 74.9%, and a Matthews Correlation Coefficient value of 0.49. Furthermore, the model also consistently provides the effective performance in independent testing set, yielding sensitivity of 75.7% and specificity of 64.9%. Finally, the model has been implemented as a web-based system, namely iDPGK, which is now freely available at http://mer.hc.mmh.org.tw/iDPGK/.

scholarly journals Phospho-islands and the evolution of phosphorylated amino acids in mammals

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10436
Author(s):  
Mikhail Moldovan ◽  
Mikhail S. Gelfand
Keyword(s):  
Amino Acids ◽  
Protein Function ◽  
Chemical Properties ◽  
Evolutionary Analysis ◽  
Post Translational Modification ◽  
Evolutionary Patterns ◽  
Charged Amino Acids ◽  
Physico Chemical ◽  
Frequent Mutations ◽  
Evolutionary Aspects

Background Protein phosphorylation is the best studied post-translational modification strongly influencing protein function. Phosphorylated amino acids not only differ in physico-chemical properties from non-phosphorylated counterparts, but also exhibit different evolutionary patterns, tending to mutate to and originate from negatively charged amino acids (NCAs). The distribution of phosphosites along protein sequences is non-uniform, as phosphosites tend to cluster, forming so-called phospho-islands. Methods Here, we have developed a hidden Markov model-based procedure for the identification of phospho-islands and studied the properties of the obtained phosphorylation clusters. To check robustness of evolutionary analysis, we consider different models for the reconstructions of ancestral phosphorylation states. Results Clustered phosphosites differ from individual phosphosites in several functional and evolutionary aspects including underrepresentation of phosphotyrosines, higher conservation, more frequent mutations to NCAs. The spectrum of tissues, frequencies of specific phosphorylation contexts, and mutational patterns observed near clustered sites also are different.

Arginine (R) at Position 595 in FLT3 Is Critical for Transforming Potential of FLT3-Length Mutations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1598-1598
Author(s):  
Sridhar Vempati ◽  
Ruth Schwab ◽  
Theodora Malamoussi ◽  
Martin Dugas ◽  
Gudrun Mellert ◽  
...  
Keyword(s):  
Amino Acids ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Sequence Motif ◽  
Acute Leukemias ◽  
Activating Mutations ◽  
Juxtamembrane Region ◽  
Length Mutations ◽  
Tandem Duplications

Abstract Activating mutations in the juxtamembrane region of FMS-like tyrosine kinase-3 (FLT3-length mutations, FLT3-LM)-are found in 20–25% of patients with acute myeloid leukemia (AML). FLT3-LM consists of duplications and additional insertions that show a variable length and site of insertion. Although these different length mutations lead to constitutive activation of FLT3 and subsequent downstream signalling pathways, several questions still remain unanswered. Here, we focussed on the question whether a common sequence motif in the duplicated region can be identified in patients carrying FLT3-LM. To address this topic we sequenced the juxtamembrane region of FLT3 from 274 patients with acute leukemias. We found that the length of mutation (tandem duplications) varied from 2–42 amino acids with a median of 17 AA. Furthermore, the duplicated amino acids centered around the motif DFREYEY of FLT3. Since the length of the motif DFREYEY varied from patient to patient, we focussed our study on the frequency of single amino acids within the duplicated region. We found that arginine (R) 595 in the motif DFREYEY is duplicated in 77% of patients. Further studies showed that a single duplication of arginine at position 595 in FLT3 is able to confer factor independent growth to Ba/F3 cells. In vitro, deletion or substitution of duplicated R 595 in two FLT3-LMs with different lengths showed a 50% reduction in the factor independent growth when compared to undeleted/non-substituted FLT3-LMs. The reduced proliferative capacity of Ba/F3 cells expressing FLT3-LM with deletions of R-595 was associated with a reduction of STAT5 activation. Our data provide important insights into the molecular mechanisms of transformation by FLT3-LM and define duplicated R 595 as a critical mediator of the leukemic potential of these mutants.

scholarly journals Allowed N-glycosylation sites on the Kv1.2 potassium channel S1–S2 linker: implications for linker secondary structure and the glycosylation effect on channel function

2003 ◽  
Vol 375 (3) ◽  
pp. 769-775 ◽  
Author(s):  
Jing ZHU ◽  
Itaru WATANABE ◽  
Amanda POHOLEK ◽  
Matthew KOSS ◽  
Barbara GOMEZ ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Glycosylation Site ◽  
Short Length ◽  
Loop Structure ◽  
Membrane Domains ◽  
Middle Section ◽  
Post Translational Modification ◽  
K Channels ◽  
Glycosylation Sites

N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51–66]. Kv1 channel S1–S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2 S1–S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of the S1–S2 linker was accessible to the endoplasmic reticulum glycotransferase at every position and was in the extracellular aqueous phase, and presumably in a flexible conformation. We speculate that the S1–S2 linker is mostly a coiled-loop structure and that the strict relative position of native glycosylation sites on these linkers may be involved in the mechanism underlying the functional effects of glycosylation on some Kv1 K+ channels. The S3–S4 linker, with 16 amino acids and no N-glycosylation site, was not glycosylated when an N-glycosylation site was added. However, an extended linker, with an added N-linked site, was glycosylated, which suggested that the native linker was not glycosylated due to its short length. Thus other ion channels or membrane proteins may also have a high glycosylation potential on a linker but yet have similarly positioned native N-glycosylation sites among isoforms. This may imply that the native position of the N-glycosylation site may be important if the carbohydrate tree plays a role in the folding, stability, trafficking and/or function of the protein.

scholarly journals A unifying mechanism for the biogenesis of prokaryotic membrane proteins co-operatively integrated by the Sec and Tat pathways

2017 ◽  
Author(s):  
Fiona J. Tooke ◽  
Marion Babot ◽  
Govind Chandra ◽  
Grant Buchanan ◽  
Tracy Palmer
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Streptomyces Coelicolor ◽  
Bioinformatic Analysis ◽  
Protein Families ◽  
Iron Sulfur Cluster ◽  
Sec Pathway ◽  
Charged Amino Acids ◽  
Loop Region ◽  
Twin Arginine Translocase

AbstractThe vast majority of polytopic membrane proteins are inserted into the cytoplasmic membrane of prokaryotes by the general secretory (Sec) pathway. However, a subset of monotopic proteins that contain non-covalently-bound redox cofactors depend on the twin-arginine translocase (Tat) machinery for membrane integration. Recently actinobacterial Rieske iron-sulfur cluster-containing proteins were identified as an unusual class of membrane proteins that require both the Sec and Tat pathways for the insertion of their three transmembrane domains (TMDs). The Sec pathway inserts the first two TMDs of these proteins co-translationally, but releases the polypeptide prior to the integration of TMD3 to allow folding of the cofactor-containing domain and its translocation by Tat. Here we have investigated features of the Streptomyces coelicolor Rieske polypeptide that modulate its interaction with the Sec and Tat machineries. Mutagenesis of a highly conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 shows that it plays no significant role in coordinating the activities of the two translocases, but that a minimum loop length of approximately eight amino acids is required for the Tat machinery to recognise TMD3. Instead we show that a combination of relatively low hydrophobicity of TMD3, coupled with the presence of C-terminal positively-charged amino acids, results in abortive insertion of TMD3 by the Sec pathway and its release at the cytoplasmic side of the membrane. Bioinformatic analysis identified two further families of polytopic membrane proteins that share features of dual Sec-Tat-targeted membrane proteins. A predicted heme-molybdenum cofactor-containing protein with five TMDs, and a polyferredoxin also with five predicted TMDs, are encoded across bacterial and archaeal genomes. We demonstrate that membrane insertion of representatives of each of these newly-identified protein families is dependent on more than one protein translocase, with the Tat machinery recognising TMD5. Importantly, the combination of low hydrophobicity of the final TMD and the presence of multiple C-terminal positive charges that serve as critical Sec-release features for the actinobacterial Rieske protein also dictate Sec release in these further protein families. Therefore we conclude that a simple unifying mechanism governs the assembly of dual targeted membrane proteins.

scholarly journals iDPGK: Characterization and Identification of Lysine Phosphoglycerylation Sites Based on Sequence-Based Features

2020 ◽  
Author(s):  
Kai-Yao Huang ◽  
Fang-Yu Hung ◽  
Hui-Ju Kao ◽  
Hui-Hsuan Lau ◽  
Shun-Long Weng
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Training Dataset ◽  
Post Translational Modification ◽  
Web Based ◽  
Charged Amino Acids ◽  
Pair Composition ◽  
Weighted Matrix ◽  
Svm Model ◽  
Effective Performance

Abstract Background:Protein phosphoglycerylation, the addition of a 1,3-bisphosphoglyceric acid (1,3-BPG) to a lysine residue of a protein and thus to form a 3-phosphoglyceryl-lysine (pgK), is a reversible and non-enzymatic post-translational modification (PTM) and plays a regulatory role in glucose metabolism and glycolytic process. As the number of experimentally verified phosphoglycerylated sites has increased significantly, statistical or machine learning methods are imperative for investigating the characteristics of phosphoglycerylation sites. Currently, research into phosphoglycerylation is very limited, and only a few resources are available for the computational identification of phosphoglycerylation sites. Result: We present a bioinformatics investigation of phosphoglycerylation sites based on sequence-based features. The TwoSampleLogo analysis reveals that the regions surrounding the phosphoglycerylation sites contain a high relatively of positively charged amino acids, especially in the upstream flanking region. Additionally, the non-polar and aliphatic amino acids are more abundant surrounding phosphoglycerylated lysine following the results of PTM-Logo, which may play a functional role in discriminating between phosphoglycerylation and non- phosphoglycerylation sites. Many types of features were adopted to build the prediction model on the training dataset, including amino acid composition, amino acid pair composition, positional weighted matrix and position-specific scoring matrix. Further, to improve the predictive power, numerous top features ranked by F-score were considered as the final combination for classification, and thus the predictive models were trained using DT, RF and SVM classifiers. Evaluation by five-fold cross-validation showed that the selected features was most effective in discriminating between phosphoglycerylated and non- phosphoglycerylated sites.Conclusion: The SVM model trained with the selected sequence-based features performed well, with a sensitivity of 77.5%, a specificity of 73.6%, an accuracy of 74.9%, and a Matthews Correlation Coefficient value of 0.49. Furthermore, the model also consistently provides the effective performance in independent testing set, yielding sensitivity of 75.7% and specificity of 64.9%. Finally, the model has been implemented as a web-based system, namely iDPGK, which is now freely available at http://mer.hc.mmh.org.tw/iDPGK/.

scholarly journals A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

2015 ◽  
Vol 112 (47) ◽  
pp. 14623-14628 ◽  
Author(s):  
Christoffer K. Goth ◽  
Adnan Halim ◽  
Sumeet A. Khetarpal ◽  
Daniel J. Rader ◽  
Henrik Clausen ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Ex Vivo ◽  
Ectodomain Shedding ◽  
Site Specific ◽  
Juxtamembrane Region ◽  
Tnf Α ◽  
Cell Membrane Proteins ◽  
A Disintegrin And Metalloproteinase

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

scholarly journals Phospho-islands and the evolution of phosphorylated amino acids in mammals

2020 ◽  
Author(s):  
Mikhail A. Moldovan ◽  
Mikhail S. Gelfand
Keyword(s):  
Amino Acids ◽  
Protein Function ◽  
Chemical Properties ◽  
Evolutionary Analysis ◽  
Post Translational Modification ◽  
Evolutionary Patterns ◽  
Charged Amino Acids ◽  
Physico Chemical ◽  
Frequent Mutations ◽  
Evolutionary Aspects

AbstractBackgroundProtein phosphorylation is the best studied post-translational modification strongly influencing protein function. Phosphorylated amino acids not only differ in physico-chemical properties from non-phosphorylated counterparts, but also exhibit different evolutionary patterns, tending to mutate to and originate from negatively charged amino acids. The distribution of phosphosites along protein sequences is non-uniform, as phosphosites tend to cluster, forming so-called phospho-islands.MethodsHere, we have developed an HMM-based procedure for the identification of phospho-islands and studied the properties of the obtained phosphorylation clusters. To check robustness of evolutionary analysis, we consider different models for the reconstructions of ancestral phosphorylation states.ResultsClustered phosphosites differ from individual phosphosites in several functional and evolutionary aspects including underrepresentation of phosphotyrosines, higher conservation, more frequent mutations to negatively charged amino acids. The spectrum of tissues, frequencies of specific phosphorylation contexts, and mutational patterns observed near clustered sites also are different.

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