Showing papers by "Elina Ikonen published in 2023"

Cholesterol esters form supercooled lipid droplets whose nucleation is facilitated by triacylglycerols

Abstract: Abstract Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs.

Data from Lipid Metabolic Reprogramming Extends beyond Histologic Tumor Demarcations in Operable Human Pancreatic Cancer

Abstract: <div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignancies and potentially curable only with radical surgical resection at early stages. The tumor microenvironment has been shown to be central to the development and progression of PDAC. A better understanding of how early human PDAC metabolically communicates with its environment and differs from healthy pancreas could help improve PDAC diagnosis and treatment. Here we performed deep proteomic analyses from diagnostic specimens of operable, treatment-naïve PDAC patients (<i>n</i> = 14), isolating four tissue compartments by laser-capture microdissection: PDAC lesions, tumor-adjacent but morphologically benign exocrine glands, and connective tissues neighboring each of these compartments. Protein and pathway levels were compared between compartments and with control pancreatic proteomes. Selected targets were studied immunohistochemically in the 14 patients and in additional tumor microarrays, and lipid deposition was assessed by nonlinear label-free imaging (<i>n</i> = 16). Widespread downregulation of pancreatic secretory functions was observed, which was paralleled by high cholesterol biosynthetic activity without prominent lipid storage in the neoplastic cells. Stromal compartments harbored ample blood apolipoproteins, indicating abundant microvasculature at the time of tumor removal. The features best differentiating the tumor-adjacent exocrine tissue from healthy control pancreas were defined by upregulation of proteins related to lipid transport. Importantly, histologically benign exocrine regions harbored the most significant prognostic pathways, with proteins involved in lipid transport and metabolism, such as neutral cholesteryl ester hydrolase 1, associating with shorter survival. In conclusion, this study reveals prognostic molecular changes in the exocrine tissue neighboring pancreatic cancer and identifies enhanced lipid transport and metabolism as its defining features.</p>Significance:<p>In clinically operable pancreatic cancer, regions distant from malignant cells already display proteomic changes related to lipid transport and metabolism that affect prognosis and may be pharmacologically targeted.</p></div>

One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

Abstract: Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

Intracellular Cholesterol Trafficking.

Abstract: Cholesterol is an essential lipid species of mammalian cells. Cells acquire it through synthesis in the endoplasmic reticulum (ER) and uptake from lipoprotein particles. Newly synthesized cholesterol is efficiently distributed from the ER to other organelles via lipid-binding/transfer proteins concentrated at membrane contact sites (MCSs) to reach the trans-Golgi network, endosomes, and plasma membrane. Lipoprotein-derived cholesterol is exported from the plasma membrane and endosomal compartments via a combination of vesicle/tubule-mediated membrane transport and transfer through MCSs. In this review, we provide an overview of intracellular cholesterol trafficking pathways, including cholesterol flux from the ER to other membranes, cholesterol uptake from lipoprotein donors and transport from the plasma membrane to the ER, cellular cholesterol efflux to lipoprotein acceptors, as well as lipoprotein cholesterol secretion from enterocytes, hepatocytes, and astrocytes. We also briefly discuss human diseases caused by defects in these processes and therapeutic strategies available in such conditions.