Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using and assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q aggregation and represents a target of future therapeutic avenues for HD.

Several pathogenic bacteria exploit human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for adhesion to and invasion into their host cells. CEACAM isoforms have characteristic expression patterns on epithelial, endothelial, or hematopoietic cells, providing bacteria with distinct sets of receptors on particular tissues. For example, while CEACAM1 and CEACAM6 have a wide tissue distribution, CEACAM3, CEACAM4, and CEACAM8 are uniquely expressed on primary human granulocytes, whereas CEA and CEACAM7 are limited to epithelia. By reconstitution of a CEACAM-deficient cell line with individual CEACAMs, we have analyzed the requirements for CEACAM-mediated internalization of Neisseria gonorrhoeae. Our results point to two mechanistically different uptake pathways triggered by either epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) or the granulocyte-specific CEACAM3. In particular, CEACAM3-mediated uptake critically depends on Src family protein tyrosine kinase (PTK) activity, and CEACAM3 associates with the SH2 domains of several Src PTKs. In contrast, epithelial CEACAMs require the integrity of cholesterol-rich membrane microdomains and are affected by cholesterol depletion, whereas CEACAM3-mediated uptake by transfected cells or the opsonin-independent phagocytosis by human granulocytes is not altered in the presence of cholesterol chelators. These results allow the subdivision of all human CEACAMs known to be utilized as pathogen receptors into functional groups and point to important consequences for bacterial engagement of distinct CEACAM isoforms.

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.

SummaryStaphylococcus aureus, which is a leading cause of hospital-acquired infections, binds via fibronectin to integrin 51, a process that can promote host colonization in vivo. Integrin engagement induces actin cytoskeleton rearrangements that result in the uptake of S. aureus by non-professional phagocytic cells. Interestingly, we found that fibronectin-binding S. aureus trigger the redistribution of membrane microdomain components. In particular, ganglioside GM1 and GPI-linked proteins were recruited upon integrin 1 engagement, and disruption of membrane microdomains blocked bacterial internalization. Several membrane-microdomain-associated proteins, such as flotillin-1 and flotillin-2, as well as caveolin, were recruited to sites of bacterial attachment. Whereas dominantnegative versions of flotillin-2 did not affect bacterial attachment or internalization, cells deficient for caveolin-1 (Cav1 -/-) showed increased uptake of S. aureus and other Fn-binding pathogens. Recruitment of membrane microdomains to cell-associated bacteria was unaltered in Cav1 -/-cells. However, fluorescence recovery after photobleaching (FRAP) revealed an enhanced mobility of membranemicrodomain-associated proteins in the absence of caveolin-1. Enhanced membrane microdomain mobility and increased uptake of S. aureus was repressed by expression of wild-type caveolin-1, but not caveolin-1 G83S, which harbors a point mutation in the caveolin scaffolding domain. Similarly, chemical or physical stimulation of membrane fluidity led to increased uptake of S. aureus. These results highlight a crucial role for caveolin-1 in negative regulation of membrane microdomain mobility, thereby affecting endocytosis of bacteria-engaged integrins. This process might not only limit host cell invasion by integrin-binding bacterial pathogens, but might also be physiologically relevant for integrin-mediated cell adhesion.

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.