These results demonstrate the ability to design and produce polymer-based nanodevices for the intracellular targeting of drugs, imaging agents, and other materials.

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity.

mRNA metabolism is tightly orchestrated by highly-regulated RNA Binding Proteins (RBPs) that determine mRNA fate, thereby influencing multiple cellular functions across biological contexts. Here, we review the interplay between six well-known RBPs (TTP, AUF-1, KSRP, HuR, TIA-1, and TIAR) that recognize AU-rich elements (AREs) at the 3′ untranslated regions of mRNAs, namely ARE-RBPs. Examples of the links between their cross-regulations and modulation of their targets are analyzed during mRNA processing, turnover, localization, and translational control. Furthermore, ARE recognition can be self-regulated by several factors that lead to the prevalence of one RBP over another. Consequently, we examine the factors that modulate the dynamics of those protein-RNA transient interactions to better understand the final consequences of the regulation mediated by ARE-RBPs. For instance, factors controlling the RBP isoforms, their conformational state or their post-translational modifications (PTMs) can strongly determine the fate of the protein-RNA complexes. Moreover, mRNA specific sequence and secondary structure or subtle environmental changes are also key determinants to take into account. To sum up, the whole understanding of such a fine tuned regulation is a challenge for future research and requires the integration of all the available structural and functional data by in vivo, in vitro and in silico approaches.

Cytochrome c (Cc) is a key protein in cell life (respiration) and cell death (apoptosis). On the one hand, it serves as a mitochondrial redox carrier, transferring electrons between the membrane-embedded complexes III and IV. On the other hand, it acts as a cytoplasmic apoptosis-triggering agent, forming the apoptosome with apoptosis protease-activating factor-1 (Apaf-1) and activating the caspase cascade. The two functions of cytochrome c are finely tuned by the phosphorylation of tyrosines and, in particular, those located at positions 48 and 97. However, the specific cytochrome c-phosphorylating kinase is still unknown. To study the structural and functional changes induced by tyrosine phosphorylation in cytochrome c, we studied the two phosphomimetic mutants Y48E and Y97E, in which each tyrosine residue is replaced by glutamate. Such substitutions alter both the physicochemical features and the function of each mutant compared with the native protein. Y97E is significantly less stable than the WT species, whereas Y48E not only exhibits lower values for the alkaline transition pK (a) and the midpoint redox potential, but it also impairs Apaf-1-mediated caspase activation. Altogether, these findings suggest that the specific phosphorylation of Tyr48 makes cytochrome c act as an anti-apoptotic switch.

No abstract

Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Human cytochrome c, with five tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain, and on the other hand, it serves as a cytoplasmic apoptosis-triggering agent. In a previous paper, we reported the effect of nitration on physicochemical and kinetic features of monotyrosine cytochrome c mutants. Here, we analyse the nitration-induced changes in secondary structure, thermal stability, haem environment, alkaline transition and molecular dynamics of three of such monotyrosine mutants--the so-called h-Y67, h-Y74 and h-Y97--which have four tyrosines replaced by phenylalanines and just keep the tyrosine residue giving its number to the mutant. The resulting data, along with the functional analyses of the three mutants, indicate that it is the specific nitration of solvent-exposed Tyr74 which enhances the peroxidase activity and blocks the ability of Cc to activate caspase-9, thereby preventing the apoptosis signaling pathway.

The complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc has been characterized by NMR spectroscopy. The binding constant is 16 mM ؊1 , and the lifetime of the complex is much less than 10 ms. Intermolecular pseudo-contact shifts observed for the plastocyanin amide nuclei, caused by the heme iron, as well as the chemical-shift perturbation data were used as the sole experimental restraints to determine the orientation of plastocyanin relative to cytochrome f with a precision of 1.3 Å. The data show that the hydrophobic patch surrounding tyrosine 1 in cytochrome f docks the hydrophobic patch of plastocyanin. Charge complementarities are found between the rims of the respective recognition sites of cytochrome f and plastocyanin. Significant differences in the relative orientation of both proteins are found between this complex and those previously reported for plants and Phormidium, indicating that electrostatic and hydrophobic interactions are balanced differently in these complexes.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals. Molecular & Cellular