Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

A druggable conformational switch in the c-MYC transactivation domain.

The c-MYC oncogene is activated in over 70% of all human cancers. The intrinsic disorder of the c-MYC transcription factor facilitates molecular interactions that regulate numerous biological pathways, but severely limits efforts to target its function for cancer therapy. Here, we use a reductionist strategy to characterize the dynamic and structural heterogeneity of the c-MYC protein. Using probe-based Molecular Dynamics (MD) simulations and machine learning, we identify a conformational switch in the c-MYC amino-terminal transactivation domain (termed coreMYC) that cycles between a closed, inactive, and an open, active conformation. Using the polyphenol epigallocatechin gallate (EGCG) to modulate the conformational landscape of coreMYC, we show through biophysical and cellular assays that the induction of a closed conformation impedes its interactions with the transformation/transcription domain-associated protein (TRRAP) and the TATA-box binding protein (TBP) which are essential for the transcriptional and oncogenic activities of c-MYC. Together, these findings provide insights into structure-activity relationships of c-MYC, which open avenues towards the development of shape-shifting compounds to target c-MYC as well as other disordered transcription factors for cancer treatment.

Mitochondrial Probe for Glutathione Depletion Reveals NME3 Essentiality for Mitochondrial Redox Response.

Maintenance of the mitochondrial thiol redox state is essential for cell survival. However, we lack a comprehensive understanding of the redox response to mitochondrial glutathione depletion. We developed a mitochondria-penetrating peptide, mtCDNB, to specifically deplete mitochondrial glutathione. A genome-wide CRISPR/Cas9 screen in tandem with mtCDNB treatment was employed to uncover regulators of the redox response to mitochondrial glutathione depletion. We identified nucleoside diphosphate kinase 3 (NME3) as a regulator of mitochondrial dynamics. We show that NME3 is recruited to the mitochondrial outer membrane when under redox stress. In the absence of NME3, there is impaired mitophagy, which leads to the accumulation of dysfunctional mitochondria. NME3 knockouts depleted of mitochondrial glutathione have increased mitochondrial ROS production, accumulate mtDNA lesions, and present a senescence-associated secretory phenotype. Our findings suggest a novel role for NME3 in selecting mitochondria for degradation through mitophagy under conditions of mitochondrial redox stress.