From the journals: JBC

Linking modified cysteines to cell migration. Recognizing protein tags for degradation. Disrupting C. difficile toxin production. Read about papers on these topics recently published in the Journal of Biological Chemistry.

Linking modified cysteines to cell migration

Redox signaling includes the posttranslational modification S-glutathionylation, which results in an oxidized cysteine residue with glutathione attached via a disulfide bond. In cells, over 2,000 cysteine residues receive this modification, but scientists cannot easily assess which S-glutathionylation events connect to a given biological process. For example, researchers want to identify redox signaling proteins that drive cell migration, a process already known to involve S-glutathionylated proteins that regulate embryogenesis and maintain healthy tissues but can also lead to cancer metastasis under pathological conditions.

An illustration of cancer cells. This study examined a breast cancer cell line to assess proteins involved in cell migration.

Dhanushika Kukulage from Drexel University and a team of researchers in the U.S. published a method in the Journal of Biological Chemistry to identify cell migration–related proteins with S-glutathionylated cysteines. Their platform uses proteomics, bioinformatics and biological screening. First, they used bioorthogonal chemistry and mass spectrometry to find proteins with the S-glutathionylation modification and compared this list with previously identified cell migration proteins. Next, they selected nine proteins based on activity classification for analysis in an oxidative stress–induced breast cancer cell migration assay. Expression of three of those proteins, the serine/threonine phosphatase PP2Cα, cytoskeletal reorganization protein ARHGEF7 and integrin signaling protein NISCH, increased oxidative stress and thus enhanced migration. The authors showed that this enhanced ability returned to baseline when they mutated the glutathionylated cysteine to serine, indicating a glutathionylation-dependent effect on migration.

PP2Cα expression showed a larger surge in cell migration, so the researchers chose this protein for further study. They determined that glutathionylation disrupts PP2Cα interactions with the kinases JNK, ERK1/2 and MEK4, which increases their phosphorylation levels and may enhance cell migration–related signaling.

Future studies will clarify how S-glutathionylation of ARHGEF7 and NISCH increases migration under oxidative stress. Finally, the authors emphasized the versatility of their platform; with different biological screens, researchers can investigate additional cellular processes and identify the associated S-glutathionylated proteins active in redox signaling.

Recognizing protein tags for degradation

The E3 ubiquitin ligase MARCHF6 is part of the N-degron protein degradation pathway that maintains protein quality control. MARCHF6 recognizes proteins with an N-terminal acetylation tag called Ac/N degrons and ubiquitinates them to drive proteolysis. Previously, scientists determined that MARCHF6 suppresses the iron-dependent cell death pathway, or ferroptosis, which is associated with numerous diseases, including cancer and neurodegeneration. Due to MARCHF6’s complex structure of transmembrane helices and cytoplasmic domains, scientists do not know the structural details of MARCHF6’s function.

Jihye Yang from Pohang University of Science and Technology and a group of researchers in South Korea examined the cytosolic domains of MARCHF6 and published their findings in the Journal of Biological Chemistry. The authors identified the region of MARCHF6 that recognizes acetylated N-terminal protein substrates and termed it the Ac/N domain because it distinguishes between acetylated and nonacetylated targets. The researchers also determined that MARCHF6-mediated degradation of the G-protein regulator RGS2 and the lipid droplet protein PLIN2 makes cells more susceptible to ferroptosis. They mutated the MARCHF6 Ac/N domain to determine that increased susceptibility to ferroptosis depends on MARCHF6’s ability to recognize acetylated RGS2 and PLIN2.

Future work will focus on dissecting MARCHF6’s substrate-dependent role in ferroptosis regulation. These findings could drive therapeutic developments for ferroptosis-related diseases, including cancer and cardiovascular diseases.

Disrupting C. difficile toxin production

Clostridioides difficile infects the colon and causes hundreds of thousands of infections annually in the U.S. Scientists want to find treatment options that target C. difficile’s unique pathophysiology, such as the biosynthesis of the toxins TcdA and TcdB, to avoid killing beneficial bacteria that help block pathogen colonization. Ravi Marreddy from Texas A&M University and a team of researchers in the U.S. recently published their work in the Journal of Biological Chemistry on the discovery of plant-derived compounds that inhibit toxin production but do not abolish bacterial growth. They investigated plant-derived compounds and selected the compound enoxolone, a licorice metabolite, for further study due to its potency in stopping C. difficile producing toxins.

The researchers used affinity-based proteomics and gene silencing to show that enoxolone halts toxin production by inhibiting adenine deaminase, which drives purine metabolism, and the adenosine triphosphate, or ATP, synthase, which is involved in energy production. The authors used surface plasmon resonance and isothermal titration calorimetry to show that enoxolone binds directly to the enzyme adenine deaminase. In addition, the authors used high-performance liquid chromatography to determine that enoxolone moderately reduced cellular ATP levels.

Future research will investigate how adenine deaminase and ATP synthase connect to toxin production and whether other enzymes in purine metabolism could become drug targets. However, in mice with C. difficile infection, treatment with enoxolone causes multiple side effects, so researchers will need to discover compounds related to enoxolone that mitigate these side effects to come up with possible treatments for C. difficile infections.