Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure in the activation of the apoptotic intrinsic pathway, thereby activating the caspase cascade through its interaction with Apaf-1. Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.

The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics.

Huntington’s Disease is a neurodegenerative disorder caused by a CAG expansion of the first exon of the HTT gene, resulting in an extended poly-glutamine (poly-Q) tract in the N-terminus of the protein huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood mainly due to its intrinsic flexibility and the strong compositional bias of the protein. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. The integrative analysis of the data reveals that the poly-Q tract adopts long α-helical conformations stabilized by glutamine side-chain to backbone hydrogen bonds. ¹⁹ F-NMR of site-specifically incorporated fluoro-glutamines and molecular dynamics simulations demonstrate that the mechanism propagating α-helical conformations towards the poly-Q from the upstream N17 domain is independent of the poly-Q track length. Aggregation and atomic force microscopy experiments show that the presence of long and persistent α-helices in the poly-Q tract is a stronger signature in defining the aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. The ensemble of our observations provides a structural perspective of the pathogenicity of expanded httex1 and paves the way to a deeper understanding of poly-Q related diseases.

The alternative reading frame (ARF) protein is crucial in the cellular response to oncogenic stress, being likewise the second most frequently inactivated gene in a wide spectrum of human cancers. ARF is usually sequestered in the nucleolus by the well-known oncogenic nucleophosmin (NPM) protein and is liberated in response to cell damage to exhibit its tumor-suppressor ability. However, the mechanism underlying ARF activation is unknown. Here we show that mitochondria-to-nucleus translocation of cytochrome c upon DNA damage leads to the break-off of the NPM-ARF ensemble and subsequent release of ARF from the nucleoli. Our structural and subcellular data support a molecular model in which the hemeprotein triggers the extended-to-compact conformation of NPM and competes with ARF for binding to NPM.

Intrinsically disordered regions (IDRs) are major drivers of protein functional diversification, yet the molecular features that enable the emergence of new functions within disordered sequences remain poorly understood. FCHO1 and FCHO2 are paralogous pioneer proteins of clathrin-mediated endocytosis that share a conserved domain architecture but perform distinct cellular functions and cannot compensate for each other's loss. Here we show that functional divergence between these proteins is associated with the acquisition of transient structure within their disordered regions. Using nuclear magnetic resonance spectroscopy, we identify two highly populated α-helical elements in the IDR of FCHO1 that are absent from FCHO2. One of these helices mediates FCHO1 self-association and drives intracellular assembly, whereas FCHO2 lacks this behavior. Introduction of the FCHO1 helix into FCHO2 is sufficient to confer self-association and cellular assembly, demonstrating that a transient structural element embedded within an IDR can act as a transferable functional module. Evolutionary analysis reveals that this helical propensity emerged following duplication of the ancestral FCHO gene and became progressively reinforced during evolution. Despite this divergence, the same region retains a conserved membrane-binding activity in both paralogs. Together, our findings show how acquisition of transient secondary structure within an intrinsically disordered region can generate new molecular behaviors while preserving ancestral functions, providing a mechanism for the functional specialization of paralogous proteins.