Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target.

Background: Oligomeric complexes of APP, APLP1, and APLP2 contribute to synapse formation and structure. Results: Zinc binding to the E2 domain of APP and APLPs promotes their oligomerization in the cell, most notably with APLP1. Conclusion: Extracellular zinc is a regulator for structure and function of APP and APLPs. Significance: Novel insight into how APP and APLP function is regulated at the molecular level.

The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Keywords: amyloid precursor protein, amyloid precursor-like protein, neuronal adhesion, number and brightness, zinc. Abbreviations used: AD, Alzheimer's disease; ADAM, a disintegrin and metalloproteinase; APLP, amyloid precursor-like protein; APP, amyloid precursor protein; Ab, b-amyloid peptide; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; cLSM, confocal laser scanning microscopy; HBS, HEPES-buffered saline; N&B, number and brightness; PDL, poly-D-lysine; PDMS, polydimethylsiloxane; PM, plasma membrane; RIP, regulated intramembrane proteolysis; sAPP/APLP, soluble APP/APLP; YFP, yellow fluorescent protein. 266

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) and its homologs, the APP like proteins APLP1 and APLP2, is typically a two-step process, which is initiated by ectodomain-shedding of the substrates by α- or β-secretases. Growing evidence, however, indicates that the cleavage process for APLP1 is different than for APP. Here, we describe that full-length APLP1, but not APP or APLP2, is uniquely cleaved by γ-secretase without previous ectodomain shedding. The new fragment, termed sAPLP1γ, was exclusively associated with APLP1, not APP, APLP2. We provide an exact molecular analysis showing that sAPLP1γ was uniquely generated by γ-secretase from full-length APLP1. Mass spectrometry analysis showed that the sAPLP1γ fragment and the longest Aβ-like peptide share the C-terminus. This novel mechanism of γ-secretase action is consistent with an ϵ-cut based upon the nature of the reaction in APP. We further demonstrate that the APLP1 transmembrane sequence is the critical determinant for γ-shedding and release of full-length APLP1. Moreover, the APLP1 TMS is sufficient to convert larger type-I membrane proteins like APP into direct γ-secretase substrates. Taken together, the direct cleavage of APLP1 is a novel feature of the γ-secretase prompting a re-thinking of γ-secretase activity modulation as a therapeutic strategy for Alzheimer disease.