RC1339/APRc from Rickettsia conorii Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes

Abstract

Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.

A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2

“…Firstly, we characterized the protease properties of Amuc_1434*. As Amuc_1434* contains the conserved "DTG" and "LLG" sequence motifs of the aspartic protease family [45], we measured its protease activity using hemoglobin as the substrate. We found that this enzyme could efficiently hydrolyze hemoglobin.…”

“…Given that the sequence of Amuc_1434* contains the conserved "DTG" and "LLG" motifs of the aspartic protease family [45], it is perhaps not suprising that Amuc_1434* has the aspartic protease activity. Thus, we measured its protease activity using hemoglobin as the substrate.…”

“…Considering that the sequence of Amuc_1434* contains the conserved "DTG" and "LLG" motifs of the aspartic protease family [45], We would like to investigate if Amuc_1434* could be completely inhibited by Pepstatin A. Strikingly, Amuc_1434* completely lost its activity when pepstatin A concentration was higher than 0.04 mM, indicating that Amuc_1434* belongs to the aspartic protease family ( Figure 5).…”

The Role of Microbial Aspartic Protease Enzyme in Food and Beverage Industries

“…Ratio of milk clotting to proteolytic activity is comparable with commercial fungal protease but has high thermostability Bacillus licheniformis Shows typical milk-clotting kinetics therapeutic intervention. is implies that a retropepsin type of aspartic protease enzymes is found in prokaryotes, signifying that these enzymes may represent an ancestral form of these proteases [16]. Acid protease produced by Bacillus subtilis, which is GRAS (genetically regarded as safe), is gradually replacing chymosin in cheese making, and protease produced from B. subtilis var.…”

A generic normalization method for proper quantification in untargeted proteomics screening

“…Thus, the proposed IS proves to be able to reduce the variability among 183 replicates while maintaining the ratios between experimental conditions. Most importantly, the use of 184 the IS not only proved to be beneficial in comparison to the total intensity but also when compared 185 with a set of other 11 popular normalization methods ( Figure 2b), which are able to largely decrease 186 the variability between replicates (Supplementary Figure 5 Rickettsia conorii [17], and NusA-3C-PSI which is a fusion of NusA protein from E. coli with the saposin-203 like domain of a plant aspartic protease; Supplementary Figure 6) were added in known amounts to 204 the secretomes immediately after medium collection to be used in targeted protein quantification in 205 this analysis. These two recombinant proteins were added in low amounts ( Supplementary Table 4), 206 only enough to be quantified and to reflect the usual levels of the proteins analyzed.…”