Biology subjects

Laraia, L.

Publications and source records attributed to Laraia, L..

7 recordsLinked to original sources

bioRxiv2026

The orientation of cholesterol's hydroxyl group affects its membrane dynamics and intracellular transport

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHLs key role in neuronal function. Many CHL actions are stereospecific, as shown by differences from its 3-hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol, CTL) and epiCHL (epicholestatrienol, epiCTL), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle cholesterol structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.

biophysics↗

bioRxiv2024

Endogenous and fluorescent sterols reveal the molecular basis for ligand selectivity of human sterol transporters

Sterol transport proteins (STPs) play a pivotal role in cholesterol homeostasis and therefore are essential for healthy human physiology. Despite recent advances in dissecting functions of STPs in the human cell, there is still a significant knowledge gap regarding their specific biological functions and a lack of suitable selective probes for their study. Here, we profile fluorescent steroid-based probes across ten STPs, uncovering substantial differences in their selectivity, aiding the retrospective and prospective interpretation of biological results generated with those probes. These results guided the establishment of an STP screening panel combining diverse biophysical assays, enabling the evaluation of 41 steroid-based natural products and derivatives. Combining this with a thorough structural analysis revealed the molecular basis for STP specific selectivity profiles, leading to the uncovering of several new potent and selective Aster-B inhibitors, and supporting the role of this protein in steroidogenesis.

biochemistry↗

bioRxiv2023

Identification of non-conventional small molecule degraders and stabilizers of squalene synthase

Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/543387v1_figu1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1645e05org.highwire.dtl.DTLVardef@58ad70org.highwire.dtl.DTLVardef@1e2c57aorg.highwire.dtl.DTLVardef@112fcbd_HPS_FORMAT_FIGEXP M_FIG C_FIG Squalene synthase (SQS) is an essential enzyme in the mevalonate pathway whose abundance and activity control cholesterol biosynthesis and homeostasis. Although catalytic inhibitors of SQS have been developed to attenuate cholesterol, none so far have been approved for therapeutic use. Herein we sought to develop SQS degraders using targeted protein degradation (TPD) as an approach to lower overall cellular cholesterol content. We found that KY02111, a small molecule ligand of SQS, could selectively cause SQS to degrade in a proteasome-dependent manner. In contrast, compounds based on the same scaffold linked to E3 ligase recruiting ligands led to SQS stabilization. Whole cell proteomic analysis found KY02111 to reduce only the levels of SQS, while lipidomic analysis determined that KY02111 treatment concomitantly reduced cellular cholesteryl ester content. SQS stabilizers were shown to shield SQS from its natural turnover without recruiting their matching E3 ligase. Our work shows that degradation of SQS is possible despite a challenging biological setting and lays the groundwork for future development of either SQS degrading or stabilizing probes.

cell biology↗

bioRxiv2023

Selective inhibition of OSBP blocks retrograde trafficking by inducing partial Golgi degradation

Sterol-binding proteins are important regulators of lipid homeostasis and membrane integrity; however, the discovery of selective small molecule modulators can be challenging due to structural similarities in the sterol binding domains. We report the discovery of highly potent and selective inhibitors of oxysterol binding protein (OSBP), which we term oxybipins. Sterol-containing chemical chimeras aimed at identifying new sterol binding proteins by targeted degradation, led to a significant reduction in Golgi-associated proteins. The degradation was found to occur at lysosomes, concomitant with changes in general protein glycosylation, indicating that the degradation of Golgi proteins was a downstream effect. By establishing a sterol transport protein biophysical assay panel, we discovered that the oxybipins potently inhibited OSBP, resulting in blockage of retrograde trafficking and attenuating Shiga toxin toxicity. As the oxybipins do not target any other sterol transporters tested, we advocate their use as chemical tools to study OSBP function and therapeutic relevance.

cell biology↗

bioRxiv2023

Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting the oxysterol-binding protein OSBP

Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced anti-leukemic properties via orthogonal chemical screening. Through multi-omics profiling and genome-scale CRISPR/Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate (PI4P) signaling at the endoplasmic reticulum (ER)-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.

pharmacology and toxicology↗

bioRxiv2021

Thermal proteome profiling reveals distinct target selectivity for differentially oxidized oxysterols

Oxysterols are produced physiologically by many species, however their distinct roles in regulating human (patho)physiology have not been studied systematically. The role of differing oxidation states and sites in mediating their biological functions is also unclear. As individual oxysterols have been associated with atherosclerosis, neurodegeneration and cancer, a better understanding of their protein targets would be highly valuable. To address this, we profiled three A- and B-ring oxidized sterols as well as 25-hydroxycholesterol using thermal proteome profiling (TPP), validating selected targets with the cellular thermal shift assay (CETSA) and isothermal dose response fingerprinting (ITDRF). This revealed that the site of oxidation has a profound impact on target selectivity, with each oxysterol possessing an almost unique set of target proteins. However, overall targets clustered in pathways relating to vesicular transport and lipid metabolism and trafficking, suggesting that while individual oxysterols bind to a unique set of proteins, the processes they modulate are highly interconnected.

biochemistry↗

bioRxiv2019

GLP-1R is downregulated in beta cells of NOD mice and T1D patients

Glucagon-like peptide 1 (GLP-1) is produced by L cells in the small intestine in response to ingested glucose and increases insulin release from pancreatic beta cells by activation of its cognate receptor (GLP-1R). Stimulation of this receptor also contributes to increased beta cell survival and regeneration. We have found that pancreatic beta cells from Non Obese Diabetic (NOD) mice express significantly lower levels of GLP-1R than C57BL/6 mice, leaving the NOD beta cells with an impaired response to GLP-1 stimulation. The lower expression appears to be caused by accelerated degradation of GLP-1R in the beta cells, a process that can be reversed by inhibiting trafficking to the lysosome. Importantly, our results appear to translate to the human disease since we also observed significantly lower expression of the GLP-1R in pancreatic islets from donors with type 1 diabetes. These results suggest that beta cell physiology may play a role in susceptibility to autoimmune inflammation.

physiology↗