Huntington’s disease is a neurodegenerative disease caused by an expanded polyQ stretch within Huntingtin (HTT) that renders the protein aggregation-prone, ultimately resulting in the formation of amyloid fibrils. A trimeric chaperone complex composed of Hsc70, DNAJB1 and Apg2 can suppress and reverse the aggregation of HTTExon1Q48. DNAJB1 is the rate-limiting chaperone and we have here identified and characterized the binding interface between DNAJB1 and HTTExon1Q48. DNAJB1 exhibits a HTT binding motif (HBM) in the hinge region between C-terminal domains (CTD) I and II and binds to the polyQ-adjacent proline rich domain (PRD) of soluble as well as aggregated HTT. The PRD of HTT represents an additional binding site for chaperones. Mutation of the highly conserved H244 of the HBM of DNAJB1 completely abrogates the suppression and disaggregation of HTT fibrils by the trimeric chaperone complex. Notably, this mutation does not affect the binding and remodeling of any other protein substrate, suggesting that the HBM of DNAJB1 is a specific interaction site for HTT. Overexpression of wt DNAJB1, but not of DNAJB1H244A can prevent the accumulation of HTTExon1Q97 aggregates in HEK293 cells, thus validating the biological significance of the HBM within DNAJB1.

Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.

Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS are highly N-glycosylated, but the biological functions of the glycans remain elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structure stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. MALDI-TOF MS revealed that eight asparagine sites were glycosylated with high-mannose type N-glycans. Deglycosylation of TconTS1 led to a 5-fold decrease in substrate affinity but to the same conversion rate relative to the untreated enzyme. After deglycosylation, no changes in secondary structure elements were observed in circular dichroism experiments. Molecular dynamics simulations revealed interactions between the highly flexible N-glycans and some conserved amino acids belonging to the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and promoting enzyme/substrate complex stability. The here-observed modulation of catalytic activity via the N-glycan shield may be a structure-function relationship intrinsic of several members of the TS family.

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