Identification of the cleavage sites in the α6A integrin subunit: structural requirements for cleavage and functional analysis of the uncleaved α6Aβ1 integrin

The α6A and α6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the α6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of α6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the α6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant α6A cDNAs were transfected into K562 cells. The mutant α6A integrin subunits were expressed in association with endogenous β1 at levels comparable to that of wild-type α6Aβ1. A single α6 polypeptide chain (150 kDa) was precipitated from transfectants expressing α6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded α6A subunits that were cleaved once into a heavy and a light chain, whereas α6A subunits with mutations in one of the two RK sequences were, like wild-type α6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of α6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two α6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because α6RKKG, α6GKKR and α6RGGR subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. α6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly cleaved, whereas α6A mutants with the RKKR sequence shifted to other positions in the α6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, α6A mutants with an α5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-β1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved α6Aβ1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant α6Aβ1, did not activate uncleaved α6Aβ1. Thus uncleaved α6Aβ1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of α6 is required to generate a proper conformation that enables the affinity modulation of the α6Aβ1 receptor by PMA.

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Proteolytic Cleavage of Bovine Adenovirus 3-Encoded pVIII

Journal of Virology ◽

10.1128/jvi.00211-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 5

Author(s):

Amit Gaba ◽

Lisanework Ayalew ◽

Niraj Makadiya ◽

Suresh Tikoo

Keyword(s):

Cleavage Site ◽

Virus Assembly ◽

Proteolytic Cleavage ◽

Efficient Production ◽

Cleavage Sites ◽

Bovine Adenovirus ◽

Protease Cleavage Site ◽

Protease Cleavage ◽

Protease Cleavage Sites ◽

Single Protease

ABSTRACT Proteolytic maturation involving cleavage of one nonstructural and six structural precursor proteins including pVIII by adenovirus protease is an important aspect of the adenovirus life cycle. The pVIII encoded by bovine adenovirus 3 (BAdV-3) is a protein of 216 amino acids and contains two potential protease cleavage sites. Here, we report that BAdV-3 pVIII is cleaved by adenovirus protease at both potential consensus protease cleavage sites. Usage of at least one cleavage site appears essential for the production of progeny BAdV-3 virions as glycine-to-alanine mutation of both protease cleavage sites appears lethal for the production of progeny virions. However, mutation of a single protease cleavage site of BAdV-3 pVIII significantly affects the efficient production of infectious progeny virions. Further analysis revealed no significant defect in endosome escape, genome replication, capsid formation, and virus assembly. Interestingly, cleavage of pVIII at both potential cleavage sites appears essential for the production of stable BAdV-3 virions as BAdV-3 expressing pVIII containing a glycine-to-alanine mutation of either of the potential cleavage sites is thermolabile, and this mutation leads to the production of noninfectious virions. IMPORTANCE Here, we demonstrated that the BAdV-3 adenovirus protease cleaves BAdV-3 pVIII at both potential protease cleavage sites. Although cleavage of pVIII at one of the two adenoviral protease cleavage sites is required for the production of progeny virions, the mutation of a single cleavage site of pVIII affects the efficient production of infectious progeny virions. Further analysis indicated that the mutation of a single protease cleavage site (glycine to alanine) of pVIII produces thermolabile virions, which leads to the production of noninfectious virions with disrupted capsids. We thus provide evidence about the requirement of proteolytic cleavage of pVIII for production of infectious progeny virions. We feel that our study has significantly advanced the understanding of the requirement of adenovirus protease cleavage of pVIII.

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Twenty years of bioinformatics research for protease-specific substrate and cleavage site prediction: a comprehensive revisit and benchmarking of existing methods

Briefings in Bioinformatics ◽

10.1093/bib/bby077 ◽

2018 ◽

Vol 20 (6) ◽

pp. 2150-2166 ◽

Cited By ~ 24

Author(s):

Fuyi Li ◽

Yanan Wang ◽

Chen Li ◽

Tatiana T Marquez-Lago ◽

André Leier ◽

...

Keyword(s):

Cleavage Site ◽

Scoring Function ◽

Proteolytic Cleavage ◽

Prediction Performance ◽

Specific Substrate ◽

Cleavage Sites ◽

Cell Cycle Protein ◽

Computational Approaches ◽

Site Prediction ◽

Cleavage Site Prediction

Abstract The roles of proteolytic cleavage have been intensively investigated and discussed during the past two decades. This irreversible chemical process has been frequently reported to influence a number of crucial biological processes (BPs), such as cell cycle, protein regulation and inflammation. A number of advanced studies have been published aiming at deciphering the mechanisms of proteolytic cleavage. Given its significance and the large number of functionally enriched substrates targeted by specific proteases, many computational approaches have been established for accurate prediction of protease-specific substrates and their cleavage sites. Consequently, there is an urgent need to systematically assess the state-of-the-art computational approaches for protease-specific cleavage site prediction to further advance the existing methodologies and to improve the prediction performance. With this goal in mind, in this article, we carefully evaluated a total of 19 computational methods (including 8 scoring function-based methods and 11 machine learning-based methods) in terms of their underlying algorithm, calculated features, performance evaluation and software usability. Then, extensive independent tests were performed to assess the robustness and scalability of the reviewed methods using our carefully prepared independent test data sets with 3641 cleavage sites (specific to 10 proteases). The comparative experimental results demonstrate that PROSPERous is the most accurate generic method for predicting eight protease-specific cleavage sites, while GPS-CCD and LabCaS outperformed other predictors for calpain-specific cleavage sites. Based on our review, we then outlined some potential ways to improve the prediction performance and ease the computational burden by applying ensemble learning, deep learning, positive unlabeled learning and parallel and distributed computing techniques. We anticipate that our study will serve as a practical and useful guide for interested readers to further advance next-generation bioinformatics tools for protease-specific cleavage site prediction.

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The two phenylalanines in the GFFKR motif of the integrin α6A subunit are essential for heterodimerization

Biochemical Journal ◽

10.1042/bj3280529 ◽

1997 ◽

Vol 328 (2) ◽

pp. 529-537 ◽

Cited By ~ 22

Author(s):

A. Annemieke DE MELKER ◽

Duco KRAMER ◽

Ingrid KUIKMAN ◽

Arnoud SONNENBERG

Keyword(s):

Point Mutations ◽

K562 Cells ◽

Cytoplasmic Domain ◽

Amino Acid Residues ◽

Wild Type ◽

Α Subunit ◽

Focal Contacts ◽

Arginine Residues ◽

A Site ◽

Laminin 1

The membrane-proximal domain of the integrin α subunit contains a conserved motif of five amino acid residues, GFFKR. We deleted this motif from the human α6A subunit and found that in COS-7 cells this mutant cannot associate with the β1 subunit and is retained in the endoplasmic reticulum. Point mutations in the GFFKR motif of the glycine residue or the two highly charged amino acids, or deletion of the lysine and arginine residues, had no effect on the ability of α6 to interact with β1 and to be expressed at the cell surface. In contrast, by replacing either of the two phenylalanines with alanine, or by deletion of both of these residues, α6 was incapable of associating with β1. The α6 point mutants that associated with β1 were expressed in K562 cells and their responsiveness to integrin-activating factors was determined. None of these transfectants bound spontaneously to laminin-1, but binding could be induced by either PMA or the stimulating anti-β1 antibody TS2/16 to the same extent as that of the wild-type transfectant. The ability of these mutants to initiate focal-contact formation in CHO cells plated on laminin-1 substrates also appeared to be unaltered. Thus the behaviour of α6 mutants involving the glycine, lysine or arginine residues was indistinguishable from that of wild-type α6 both in inside-out and outside-in signalling. In contrast, deletion of the cytoplasmic domain of α6 C-terminal of the GFFKR motif resulted in a loss of responsiveness of α6β1 to PMA stimulation and formation of focal contacts on laminin-1. However, this mutant was targeted to focal contacts formed by other integrins, even when they had not bound ligand. Together, these results suggest that the two phenylalanine residues of the GFFKR motif provide a site for interaction of the α6A subunit with β1, whereas the cytoplasmic domain C-terminal of this motif is involved in the regulation of bidirectional signalling via α6Aβ1.

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SARS-CoV-2 and MERS-CoV Share the Furin Site CGG-CGG Genetic Footprint

10.20944/preprints202110.0080.v2 ◽

2021 ◽

Author(s):

Antonio R. Romeu

Keyword(s):

Middle East ◽

Markov Model ◽

Cleavage Site ◽

Spike Protein ◽

Cleavage Sites ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Arginine Residues ◽

Codon Pair ◽

Shed Light

The SARS-CoV-2 polybasic furin cleavage site is still a missing link. Remarkably, the two arginine residues of this protease recognition site are encoded by the CGG codon, which is rare in Betacoronavirus. However, the arginine pair is common at viral furin cleavage sites, but are not CGG-CGG encoded. The question is: Is this genetic footprint unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers of the Betacoronavirus proteins. The main result reveals that a group of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) also have the CGG-CGG arginine pair in the spike protein polybasic furin cleavage region. In addition, CGG-CGG encoded arginine pairs were found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the probability of finding the arginine CGG-CGG codon pair in Betacoronavirus, the likelihood ratio (LR) and a Markov model were defined. In conclusion, it is highly unlikely to find this genetic marker in betacoronaviruses wildlife, but they are there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine pair in the S1/S2 cleavage site.

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TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage

Blood ◽

10.1182/blood-2014-04-569640 ◽

2014 ◽

Vol 124 (26) ◽

pp. e49-e60 ◽

Cited By ~ 36

Author(s):

Anna Prudova ◽

Katherine Serrano ◽

Ulrich Eckhard ◽

Nikolaus Fortelny ◽

Dana V. Devine ◽

...

Keyword(s):

Human Platelet ◽

Proteolytic Cleavage ◽

Human Platelets ◽

Proteolytic Processing ◽

Cleavage Sites ◽

Key Points ◽

Platelet Proteins

Key Points TAILS proteomics identified 2938 human platelet proteins, pervasive proteolytic processing, and precise proteolytic cleavage sites in stored platelets. During storage, metalloproteinases were predominantly involved in proteolytic processing, while other proteinases were mainly involved in degradation.

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Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis

Biochemical Journal ◽

10.1042/bj2260853 ◽

1985 ◽

Vol 226 (3) ◽

pp. 853-858 ◽

Cited By ~ 1

Author(s):

R J Ferl

Keyword(s):

Alcohol Dehydrogenase ◽

Sodium Dodecyl Sulphate ◽

Cleavage Site ◽

Sodium Dodecyl ◽

Protein Molecule ◽

Cleavage Sites ◽

Wild Type ◽

Induced Mutants ◽

Digestion Pattern ◽

Alcohol Dehydrogenase 1

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.

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Identification of Proteolytic Cleavage Sites in the Conversion of Profilaggrin to Filaggrin in Mammalian Epidermis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)94264-x ◽

1989 ◽

Vol 264 (3) ◽

pp. 1837-1845 ◽

Cited By ~ 3

Author(s):

K A Resing ◽

K A Walsh ◽

J Haugen-Scofield ◽

B A Dale

Keyword(s):

Proteolytic Cleavage ◽

Cleavage Sites

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A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting

Scientific Reports ◽

10.1038/s41598-021-92647-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yağmur Demircan Yalçın ◽

Taylan Berkin Töral ◽

Sertan Sukas ◽

Ender Yıldırım ◽

Özge Zorlu ◽

...

Keyword(s):

Drug Resistance ◽

K562 Cells ◽

Leukemia Cells ◽

Drug Resistant ◽

Wild Type ◽

Gene Expressions ◽

Before And After ◽

The Difference ◽

Selection Of ◽

Resistant Cells

AbstractWe report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.

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