Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering.

A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1.

Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration.

Glioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative growth of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts revealed a significantly higher expression of Glycosyltransferase 8 domain containing 1 (GLT8D1) compared to normal brain tissue and could be associated with impaired patient survival. Increased in vitro expression of GLT8D1 significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in the predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration.