Functional Study of the Retrotransposon-Derived Human PEG10 Protease

Golda, Mária; Mótyán, János András; Mahdi, Mohamed; Tőzsér, József

doi:10.3390/ijms21072424

Cited by 16 publications

(15 citation statements)

References 49 publications

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“…The overall structural characteristics imply higher similarity with retroviral-like proteases rather with retroviral proteases, e.g., HIV-1 PR. In agreement with this, the above mentioned features highly resemble those of predicted for the Saccharomyces cerevisiae retrotransposon Ty1 protease [37] and for the human retrotransposon-derived paternally expressed gene 10 (PEG10) retroviral-like protease [50], which implies similar structural characteristics for eukaryotic retroviral-like proteases.…”

Section: Discussionsupporting

confidence: 78%

“…Similarly to the retrotransposon Ty1 protease [ 37 ] and the human PEG10 protease [ 50 ], SASP14 was also found to have a natural resistance against multiple clinical protease inhibitors. Although, we compared the sequence of SASP14 to the sequences of those HIV-1 PR variants which are resistant against various protease inhibitors ( Figure 9 ), the resistance development cannot be interpreted exclusively at the level of resistance residues in equivalent positions, because the similarities and differences of the whole sequences (including active site composition) and structures need also to be considered.…”

Section: Discussionmentioning

confidence: 99%

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Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

et al. 2020

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The human retroviral-like aspartic protease 1 (ASPRV1) is a mammalian retroviral-like enzyme that catalyzes a critical proteolytic step during epidermal differentiation; therefore, it is also referred to as skin-specific aspartic protease (SASPase). Neutrophil granulocytes were also found recently to express ASPRV1 that is involved in the progression of acute chronic inflammation of the central nervous system, especially in autoimmune encephalomyelitis. Thus, investigation of ASPRV1 is important due to its therapeutic or diagnostic potential. We investigated the structural characteristics of ASPRV1 by homology modeling; analysis of the proposed structure was used for interpretation of in vitro specificity studies. For in-vitro characterization, activities of SASP28 and SASP14 enzyme forms were measured using synthetic oligopeptide substrates. We demonstrated that self-processing of SASP28 precursor causes autoactivation of the protease. The highest activity was measured for GST-SASP14 at neutral pH and at high ionic strength, and we proved that pepstatin A and acetyl-pepstatin can also inhibit the protease. In agreement with the structural characteristics, the relatively lower urea dissociation constant implied lower dimer stability of SASP14 compared to that of HIV-1 protease. The obtained structural and biochemical characteristics support better understanding of ASPRV1 function in the skin and central nervous system.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

et al. 2020

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“…A His 6 -MBP-TSAVLQ*SGFRKM-mEYFP recombinant substrate—containing a natural cleavage site of SARS-CoV-2 polyprotein—was designed and used as a positive control in cleavage reactions. This substrate system has already been applied previously to study proteases of HIV-1, tobacco etch virus [ 33 , 34 , 35 ], yeast Ty1 retrotransposon [ 36 ], and Venezuelan equine encephalitis virus (VEEV) [ 37 ], and the protease of human paternally expressed gene 10 (PEG10) protein [ 38 ]. The successful adaptation of this recombinant substrate system enables enzymatic characterization of SARS-CoV-2 3CLpro and screening of inhibitors using a microcentrifuge tube- [ 33 , 36 ] or a microtiter plate-based protease assay [ 34 , 37 ] in the future.…”

Section: Discussionmentioning

confidence: 99%

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

Miczi

Golda

Kunkli

et al. 2020

IJMS

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The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.

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“…The enzyme activity measurements were performed by using a recombinant fluorescent protein substrate-based protease assay, which has been previously applied successfully to study TEV and HIV-1 proteases [ 23 ], the retroviral-like protease of human paternally expressed gene 10 (PEG10) protein [ 35 ] and yeast retrotransposon Ty1 [ 36 ]. The previously developed method was slightly modified and some improvements were introduced as follows.…”

Section: Discussionmentioning

confidence: 99%

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates

Bozóki

Mótyán

Hoffka

et al. 2020

IJMS

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The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1′, and P2′ variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1′ amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.

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Functional Study of the Retrotransposon-Derived Human PEG10 Protease

Cited by 16 publications

References 49 publications

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates

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