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Biomedical subjects

Robert Ahrends

Publications and source records attributed to Robert Ahrends.

3 recordsLinked to original sources

High-Fat Diet and a High Amyloid Load Interact to Induce PKC-α Dependent Synaptic Insulin Resistance.

A plethora of studies suggest that a high-fat diet in combination with a high amyloid load causes synaptic insulin resistance and is a risk factor for Alzheimer's disease. Our understanding of the underlying mechanisms is still fragmented. To gain new insights, we conducted integrated proteomic and phosphoproteomic profiling of hippocampal synaptosomes from WT and a transgenic mouse line with a high amyloid load (heterozygous TBA2.1 mice) that show no overt signs of neurodegeneration and dementia. Mice were fed with a regular or high-fat diet. Data-independent acquisition quantified over 5400 proteins, revealing a stable synaptic proteome across conditions. However, the combination of high amyloid load and high-fat diet triggered coordinated remodeling of lipid metabolism pathways, particularly mitochondrial and peroxisomal fatty acid catabolism. Phosphoproteomic analysis showed pronounced activation of lipid- and stress-responsive kinases, including protein kinase C-α, along with increased inhibitory phosphorylation of insulin receptor substrates (IRS1/2). In vitro experiments indicate that blocking protein kinase C-α indeed prevents synaptic insulin resistance in primary neurons. The findings suggest that this proteomic workflow, combined with kinase pathway analysis, can reveal nodal points for interventions in a complex disease state with a trajectory to Alzheimer's disease.

Animals↗

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.

Adenosine Triphosphatases↗

Mapping protein-protein interactions between MutL and MutH by cross-linking.

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.

Adenosine Triphosphatases↗