Lipidomics Profiling Reveals Differential Alterations after FAS Inhibition in 3D Colon Cancer Cell Culture Models.

Cancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB-2640 (Denifanstat) is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes a decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.

Enhancement of Lipid Signals in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with Ammonium Fluoride as a Matrix Additive.

Lipids are essential macromolecules that play a crucial role in numerous biological events. Lipids are structurally diverse which allows them to fulfill multiple functional roles. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool to understand the spatial localization of lipids within biological systems. Herein, we report the use of ammonium fluoride (NH4F) as a comatrix additive to enhance lipid detection in biological samples, with a signal increase of up to 200%. Emphasis was placed on anionic lipid enhancement with negative polarity measurements, with some preliminary work on cationic lipids detailed. We observed lipid signal enhancement of [M-H]- ions with the addition of NH4F additive attributed to a proton transfer reaction in several different lipid classes. Overall, our study demonstrates that the use of the NH4F comatrix additive substantially improves sensitivity for lipid detection in a MALDI system and is capable of being applied to a variety of different applications.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly.

Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography-mass spectrometry (LC-MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.

Reconstitution, characterization, and [2Fe-2S] cluster exchange reactivity of a holo human BOLA3 homodimer.

A new class of mitochondrial disease has been identified and characterized as Multiple Mitochondrial Dysfunctions Syndrome (MMDS). Four different forms of the disease have each been attributed to point mutations in proteins involved in iron-sulfur (Fe-S) biosynthesis; in particular, MMDS2 has been associated with the protein BOLA3. To date, this protein has been characterized in vitro concerning its ability to form heterodimeric complexes with two putative Fe-S cluster-binding partners: GLRX5 and NFU. However, BOLA3 has yet to be characterized in its own discrete holo form. Herein we describe procedures to isolate and characterize the human holo BOLA3 protein in terms of Fe-S cluster binding and trafficking and demonstrate that human BOLA3 can form a functional homodimer capable of engaging in Fe-S cluster transfer.