Huntingtin disrupts lipid bilayers in a polyQ-length dependent manner

AbstractHuntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 N-terminal amino acids of htt have been shown to further modulate aggregation. Additionally, these 17 amino acids appear to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-lengths (35Q, 46Q, 51Q, and myc-53Q) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. Furthermore, the addition of an N-terminal myc-tag to the htt exon1 fragments impeded the interaction of htt with the bilayer

Global proteomics insights for a novel small compound targeting the non-integrin Laminin Receptor in a macrophage cell model

Introduction: Monocytes and macrophages are the first barrier of the innate immune system, which interact with agents causing osteoarthritis or other conditions, leading to the release of proinflammatory mediators that exacerbate inflammation.Methods: The aim of this study was to investigate the proteomic changes in THP-1 monocytes differentiated to macrophages, pre- or -post small compound treatments and in the presence or absence of a proinflammatory stimulus, Lipopolysaccharide (LPS). This study aimed to discover and isolate small compounds that mimic the interaction between Pigment derived growth factor (PEDF) and its 37/67 kDa Laminin receptor (LR) with potential anti-inflammatory activity.Results: Our results suggested that novel compounds targeting the LR-PEDF interface can be useful for modulating anti-inflammatory effects. Several compounds were selected based on in silico docking at the PEDF/LR interface and examined for their ability to reduce IL-1β expression in a macrophage cell model. Compound C3 showed the highest efficacy in reducing IL-1β expression in the presence of LPS proinflammatory stimulus. Proteomics analysis revealed that C3 treatment altered the global proteomic profile of THP-1 activated macrophages, affecting pathways such as MYC targets, oxidative phosphorylation, and mTORC1 signaling.Discussion: The analysis also highlighted the involvement of key regulators, including RPSA and MYC, and their interactions with other proteins such as ribosome proteins and cell cycle regulators. Furthermore, the downregulated proteome analysis revealed shared and unique pathways affected by the treatments, including processes related to actin cytoskeleton, translation, and the inflammatory response. Protein-protein interaction networks suggested the potential involvement of transcription factors like MYC and the interconnectedness of signaling pathways in mediating such as the effects of the treatments. Overall, these findings provide valuable insights into the potential anti-inflammatory activity and underlying mechanisms of compound C3, emphasizing its relevance for further investigation in the context of inflammatory conditions

Targeting and Understanding the Function of Laminin Receptor

Laminin receptor (67 LR) is a 67 kDa protein derived from a 37 kDa precursor protein (37 LR) and the derivation process most likely involves post-translational modification of 37 LR, thus laminin receptor is referred to as 37/67 LR. Found in both the membrane and cytosolic compartments, 37/67 LR normally binds laminin and pathologically is associated with cancer progression, metastasis, and invasion. We used an in silico drug discovery approach to target the interaction interface between the potent anti-angiogenic protein pigment epithelium derived factor (PEDF) and 37 LR using a Maybridge HitFinder library. Following cell based counter screening and binding validation, we characterized a hit compound’s anti-viability, activation of PEDF signaling-related genes, anti-wound healing, and anti-cancer signaling properties in a PC-3 prostate cancer cell model. Our hit compound, HTS07944, which we nicknamed C3, was able to bind the laminin interacting domain of 37 LR (Peptide G), inhibited PC-3 cell viability at low micromolar concentrations, elicited PEDF-like gene expression and inhibited angiogenic tube formation and wound healing in a scratch assay. Proteomic analysis revealed that C3’s mechanism of action may be through inhibition of chromosomal maintenance proteins (MCM family and ubiquitin E2 ligases) and pathway analysis suggested that C3 would inactive the oncogene MYC. C3 inhibited MYC binding in a luciferase promoter assay. This hit compound has potential for future development as a lead compound for treating tumor growth and inhibiting angiogenesis. To better understand function of 37 LR, we designed a series of constructs lacking critical lysines in putative small ubiquitin-like modifier (SUMO) motifs of 37 LR (K11, K42, K212) and tested the hypothesis that SUMOylation generated the 67 LR species. SUMOylation is a ligase driven, lysine directed conjugation of approximately 12 kDa proteins to client proteins that alters protein-protein interactions, cellular location/trafficking, and general function. After failure to detect 67 LR, we examined the role putative SUMO motifs played in the general fate and function of 37 LR. Using phylogenetic analysis, we showed that a common consensus SUMO motif arose in mammals and via SUMO-1 pulldown and confocal analysis using anti-SUMO-2/3 antibody, we demonstrated that 37 LR associates with SUMO. Mutations to putative SUMO motifs altered half-life of 37 LR, with a marked enhancement to stability in the case of K42R mutation as shown via cyclohexamide chase assay. K42R also failed to accumulate after MG-132 treatment, suggesting that 37 LR may possess either a direct ubiquitin (Ub) site or a mixed SUMO/Ub site that regulates proteasomal degradation. Functionally, mutations to putative SUMO motifs in 37 LR significantly altered steady state levels of early pre-rRNA including 47S and 45S. Taken together, we have highlighted a role for SUMO in the function of 37 LR