Regulation of Aberrant Protein Production (RAPP) is a protein quality control in mammalian cells. RAPP degrades mRNAs of nascent proteins not able to associate with their natural interacting partners during synthesis at the ribosome. However, little is known about the molecular mechanism of the pathway, its substrates, or its specificity. The Signal Recognition Particle (SRP) is the first interacting partner for secretory proteins. It recognizes signal sequences of the nascent polypeptides when they are exposed from the ribosomal exit tunnel. Here, we reveal the generality of the RAPP pathway on the whole transcriptome level through depletion of human SRP54, an SRP subunit. This depletion triggers RAPP and leads to decreased expression of the mRNAs encoding a number of secretory and membrane proteins. The loss of SRP54 also leads to the dramatic upregulation of a specific network of HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. These results demonstrate the complex nature of defects in protein trafficking, mRNA and protein quality control, and provide better understanding of their mechanisms at the ribosome.

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.

The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain.

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate's reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

The infectious disease tuberculosis is one of the fifteen most common causes of death worldwide (according to the WHO). About every fourth person is infected with the main causative agent Mycobacterium tuberculosis (Mb). A characteristic of the pathogen is its entrance into a dormant state in which a phenotypic antibiotic resistance is achieved. To target resistant strains, novel dormancy-specific targets are very promising. Such a possible target is the Mb "fatty acid-CoA ligase 6" (MbFACL6), which activates fatty acids and thereby modulates the accumulation of triacylglycerol-containing lipid droplets that are used by Mb as an energy source during dormancy. We investigated the membrane association of MbFACL6 in E. coli and its specific activity towards different substrates after establishing a novel MbFACL6 activity assay. Despite a high homology to the mammalian family of fatty acid transport proteins, which are typically transmembrane proteins, our results indicate that MbFACL6 is a peripheral membrane-attached protein. Furthermore, MbFACL6 tolerates a broad spectrum of substrates including saturated and unsaturated fatty acids (C12-C20), some cholic acid derivatives, and even synthetic fatty acids, such as 9(E)-nitrooleicacid. Therefore, the substrate selectivity of MbFACL6 appears to be much broader than previously assumed.

Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain, with propensity to adopt α-helical structure, interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system.

p53 exerts its tumour suppressor activity by modulating hundreds of genes and it can also repress viral replication. Such is the case of human papillomavirus (HPV) through targeting the E2 master regulator, but the biochemical mechanism is not known. We show that the C-terminal DNA binding domain of HPV16 E2 protein (E2C) triggers heterotypic condensation with p53 at a precise 2/1 E2C/p53 stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Interestingly, transfection experiments show that E2 co-localizes with p53 in the nucleus with a grainy pattern, and recruits p53 to chromatin-associated foci, a function independent of the DNA binding capacity of p53 as judged by a DNA binding impaired mutant. Depending on the length, DNA can either completely dissolve or reshape heterotypic droplets into irregular condensates containing p53, E2C, and DNA, and reminiscent of that observed linked to chromatin. We propose that p53 is a scaffold for condensation in line with its structural and functional features, in particular as a promiscuous hub that binds multiple cellular proteins. E2 appears as both client and modulator, likely based on its homodimeric DNA binding nature. Our results, in line with the known role of condensation in eukaryotic gene enhancement and silencing, point at biomolecular condensation of E2 with p53 as a means to modulate HPV gene function, strictly dependent on host cell replication and transcription machinery.