Signal peptide peptidase (SPP) and SPP‐like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II‐oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER‐resident family members SPP and SPPL2c were shown to also cleave tail‐anchored proteins, including selected SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co‐expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail‐anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1‐4 levels in tissues and primary cells of SPPL2a/b double‐deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1‐4 in a cell type‐ and tissue‐dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1‐4 by initiating the degradation of these proteins, which might impact cellular trafficking.
Recent developments in water resource monitoring have increased the demand for the reliable identification of faecal pollution sources, also defined as microbial (faecal) source tracking (MST). Standardized faecal indicator bacteria (SFIB) enumeration does not directly support MST, as SFIB occur in animal and human sources. The aim of this study was to rigorously evaluate the applicability of host-associated faecal genetic MST markers detected by quantitative PCR (qPCR) at representative Austrian water resources (ground-, surface-, raw and treated wastewater, n = 196 samples) with high importance for the water management sector. Groundwater covered a gradient of non- (i.e., deep wells) to surface influenced resources (i.e., karst and shallow wells). In addition, single faecal excreta from humans as well as representative livestock and wildlife species were collected to evaluate the faecal source-specificity and -sensitivity of the MST assays. Genetic MST marker resistance against UV irradiation was evaluated in on-site ground and wastewater treatment installations. Bacteroides-based human- (HF183II, BacHum), ruminant- (BacR), and pig-associated (Pig2Bac) MST marker qPCR quantification was performed in concert with cultivation of E. coli, intestinal enterococci, and Clostridium perfringens (SFIB diagnostics). The selected MST makers revealed high faecal source identification capacity for the Austrian water compartments and quantitatively reflected the selected faecal pollution gradient. The study also demonstrated that SFIB data can efficiently be combined with MST data to solve previously unanswered questions in water safety monitoring and management (e.g., support pollution source-targeted catchment protection, hazard assessment, and health risk management). Further research and development needs are discussed to exploit the full power of MST technology. In conclusion, this study illustrates the capacity of molecular faecal pollution diagnostics to revolutionize water quality testing in the decades to come.
N,N,N’,N’-Tetramethyl-formamidinium chloride (2a) reacts with elemental sodium in various solvents to give N,N,N’,N’,N’’,N’’-hexamethyl-guanidinium chloride (4a). The reaction of 2a with potassium affords N,N,N’,N’,N’’,N’’,N’’’,N’’’-octamethyl-oxamidinium dichloride (3a). From the reaction of 2a with magnesium in different solvents in general result mixtures of the salts 4a, 3a and N,N,N’,N’-tetramethyl-formamidinium chloride (10a). The composition of these mixtures depends on the solvent and the reaction temperature. Similar results are obtained, when a zinc/copper couple is used instead of magnesium. Very likely from 2a and magnesium or zinc, respectively, organometallic intermediates 11, 12 are formed which could be trapped by aromatic aldehydes and phenylisocyanate. The salt 2a can be reductively coupled by a low-valent titanium reagent to give the oxamidinium salt 3a.
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