Pitter Huesgen

Proteolysis is an irreversible post-translational modification that regulates many intra- and intercellular processes, including essential go/no-go decisions during cell proliferation, development and cell death. Hundreds of protease-coding genes have been identified in plants, but few have been linked to specific substrates. Conversely, proteolytic processes are frequently observed in plant biology but rarely have they been ascribed to specific proteases. In mammalian systems, unbiased system-wide proteomics analyses of protease activities have recently been tremendously successful in the identification of protease substrate repertoires, also known as substrate degradomes. Knowledge of the substrate degradome is key to understand the role of proteases in vivo. Quantitative shotgun proteomic studies have been successful in identifying protease substrates, but while simple to perform they are biased toward abundant proteins and do not reveal precise cleavage sites. Current degradomic...

Organisms that perform oxygenic photosynthesis are subjected to photoinhibition of their photosynthetic function when exposed to excessive illumination. The main target of photoinhibition is the D1 protein in the reaction center of the photosystem II complex. Rapid degradation of photodamaged D1 protein and its replacement by a de novo synthesized functional copy represent an important repair mechanism crucial for cell survival under light stress conditions. This review summarizes the literature on the ATP-independent Deg/HtrA family of serine endopeptidases in cyanobacteria and chloroplasts of higher plants, and discusses their role in D1 protein degradation. We propose that Deg/HtrA proteases are part of a larger network of enzymes that ensure protein quality control, including photosystem II, in plants and cyanobacteria.

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocy...

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.

Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and macromolecular substrates and indicates that their hydrolytic activity is influenced by the amino acid sequences adjacent to the scissile bond.

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 activit...