Summary Eukaryotic RNases H2 consist of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi-Goutières syndrome. To examine whether these mutations affect complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding three subunits were co-expressed in E. coli and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced as compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII (Tk-RNase HII). To analyze the role of this residue, four mutant proteins Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P were constructed. All mutant proteins were less stable than the wild-type protein by 2.9–7.6°C in Tm. Comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggest that introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. The findings that the mutations in the accessory subunits of Sc-RNase H2* do not seriously affect the enzymatic activity suggest that the mutant forms of the protein are relatively unstable or interactions with other proteins are perturbed in human cells.
Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T m and 12.5 kJ mol (-1) in Delta G(H 2O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T m and 1.7-5.2 kJ mol (-1) in Delta G(H 2O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T m and 12.2 kJ mol (-1) in Delta G(H 2O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.
Psychrophiles and psychrotrophs are defined as microorganisms that can grow even at around 0°C [1]. Enzymes from these microorganisms are usually less stable than those from mesophiles and thermophiles [2][3][4]. It has been reported that a decreased number of ion pairs and hydrogen bonds, decreased hydrophobic interactions and packing at the core, an increased fraction of nonpolar surface area, a decreased surface hydrophilicity, decreased helix stability and a decreased number of proline residues in the loop regions are responsible for their thermolability [5][6][7][8]. However, the destabilization mechanism of these enzymes remains to be fully understood. One promising strategy to understand this mechanism is to The Arg97 fi Gly and Asp136 fi His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7°C, respectively, in T m , and 3.5 and 6.1 kJAEmol )1 , respectively, in. These mutations also stabilized the So-RNase HI derivative (4·-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6·-RNase HI) was more stable than the wild-type protein by 28.8°C in T m and 27.0 kJAEmol )1 in DG(H 2 O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6·-RNase HI protein was determined at 2.5 Å resolution. The main chain fold and interactions of the side-chains of the 6·-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6·-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability.Abbreviations 4·-RNase HI, So-RNase HI derivative with Asn29 fi Lys, Asp39 fi Gly, Met76 fi Val and Lys90 fi Asn mutations; 5·-RNase HI, 4·-RNase HI derivative with additional Arg97 fi Gly mutation; 6·-RNase HI, 5·-RNase HI derivative with additional Asp136 fi His mutation; D136H-RNase HI, So-RNase HI derivative with Asp136 fi His mutation; Ec-RNase HI, E. coli RNase HI; GdnHCl, guanidine hydrochloride; PDB, Protein Data Bank; R97G-RNase HI, So-RNase HI derivative with Arg97 fi Gly mutation; So-RNase HI, RNase HI from Shewanella oneidensis MR-1; Tt-RNase HI, RNase HI from Thermus thermophilus.
Tujuan penelitian ini adalah mengetahui efek dan mekanisme kerja ekstrak teh Rosella merah (Hibiscus sabdariffa Linn) terhadap aktifasi NF-κβ dan ekspresi protein TNF-α serta ICAM-1 yang menjadi mediator inflamasi pada aterosklerosis. Penelitian ini menggunakan kultur sel endotel yang diisolasi dari vena umbilikalis manusia (HUVECs). Kelompok kontrol digunakan HUVECs tanpa paparan ox-LDL (kontrol negatif) dan HUVECs yang dipapar 40 µgml-1 Ox-LDL (kontrol positif). Kelompok perlakuan adalah HUVECs yang dipapar dengan berbagai dosis teh Rosella merah (0,01 mgml-1 , 0,005 mgml-1 dan 0,001 mgml-1) dan diberikan selama 2 jam sebelum dipapar ox-LDL. Pengukuran aktifasi NF-κβ dilakukan setelah 30 menit paparan Ox-LDL menggunakan imunohistokimia. Ekspresi protein TNF-α dan ICAM-1 diukur setelah 24 jam dipapar Ox-LDL menggunakan imunohistokimia. Berdasarkan analisis ANOVA (p<0.01) terdapat efek penghambatan ekstrak teh Rosella merah (Hibiscus sabdariffa Linn) terhadap aktifasi NF-κβ dan ekspresi protein TNF-α serta ICAM-1 yang manjadi mediator terjadinya inflamasi pada aterosklerosis melalui penghambatan aktifasi NF-κβ. Terdapat hubungan negatif antara aktifasi NF-κβ dan ekspresi protein TNF-α serta ICAM-1 dengan dosis ekstrak teh Rosella merah (Analisis Spearman's [p<0,01, Correlation Coeff =-1]). Kata kunci: atherosklerosis, ICAM-1, NF-κβ, Ox-LDL, Rosella merah (Hibiscus sabdariffa Linn), TNF-α PENDAHULUAN Penyakit kardiovaskuler menjadi masalah kesehatan di dunia dan di Indonesia. Kardiovaskuler juga merupakan penyebab kematian utama di dunia sampai tahun 2020, termasuk juga penyakit jantung koroner dan pembuntuan pembuluh darah otak yang diantaranya disebabkan oleh aterosklerosis [1]. Aterosklerosis merupakan proses inflamasi atau keradangan kronis yang dihasilkan sel radang. Peradangan ini dipicu oleh modifikasi Low Density Lipoprotein (LDL) yang poten sebagai penyebab aterosklerosis adalah oxidized LDL [2]. Oxidized LDL (Ox-LDL) meningkatkan ROS (Reactive Oxigen Species). Oxidized LDL bersifat sitotoksis dan berfungsi sebagai kemotaksis faktor bagi monosit yang mengakibatkan penumpukan sel-sel radang. Keradangan terjadi karena Ox-LDL mengaktifkan faktor transkripsi Nuclear Factor Kappa Beta (NF-κβ). NF-κβ yang teraktifasi akan menginduksi terbentuknya
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