Design of Glycoengineered IL-4 Antagonists Employing Chemical and Biosynthetic Glycosylation.

Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.

Ribosome-associated quality-control mechanisms from bacteria to humans.

Ribosome-associated quality-control (RQC) surveys incomplete nascent polypeptides produced by interrupted translation. Central players in RQC are the human ribosome- and tRNA-binding protein, NEMF, and its orthologs, yeast Rqc2 and bacterial RqcH, which sense large ribosomal subunits obstructed with nascent chains and then promote nascent-chain proteolysis. In canonical eukaryotic RQC, NEMF stabilizes the LTN1/Listerin E3 ligase binding to obstructed ribosomal subunits for nascent-chain ubiquitylation. Furthermore, NEMF orthologs across evolution modify nascent chains by mediating C-terminal, untemplated polypeptide elongation. In eukaryotes, this process exposes ribosome-buried nascent-chain lysines, the ubiquitin acceptor sites, to LTN1. Remarkably, in both bacteria and eukaryotes, C-terminal tails also have an extra-ribosomal function as degrons. Here, we discuss recent findings on RQC mechanisms and briefly review how ribosomal stalling is sensed upstream of RQC, including via ribosome collisions, from an evolutionary perspective. Because RQC defects impair cellular fitness and cause neurodegeneration, this knowledge provides a framework for pathway-related biology and disease studies.

Bacterial ribosome collision sensing by a MutS DNA repair ATPase paralogue.

Ribosome stalling during translation is detrimental to cellular fitness, but how this is sensed and elicits recycling of ribosomal subunits and quality control of associated mRNA and incomplete nascent chains is poorly understood1,2. Here we uncover Bacillus subtilis MutS2, a member of the conserved MutS family of ATPases that function in DNA mismatch repair3, as an unexpected ribosome-binding protein with an essential function in translational quality control. Cryo-electron microscopy analysis of affinity-purified native complexes shows that MutS2 functions in sensing collisions between stalled and translating ribosomes and suggests how ribosome collisions can serve as platforms to deploy downstream processes: MutS2 has an RNA endonuclease small MutS-related (SMR) domain, as well as an ATPase/clamp domain that is properly positioned to promote ribosomal subunit dissociation, which is a requirement both for ribosome recycling and for initiation of ribosome-associated protein quality control (RQC). Accordingly, MutS2 promotes nascent chain modification with alanine-tail degrons-an early step in RQC-in an ATPase domain-dependent manner. The relevance of these observations is underscored by evidence of strong co-occurrence of MutS2 and RQC genes across bacterial phyla. Overall, the findings demonstrate a deeply conserved role for ribosome collisions in mounting a complex response to the interruption of translation within open reading frames.

Mimicry of Canonical Translation Elongation Underlies Alanine Tail Synthesis in RQC.

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.

Quantitative basis of meiotic chromosome synapsis analyzed by electron tomography.

The synaptonemal complex is a multiprotein complex, which mediates the synapsis and recombination between homologous chromosomes during meiosis. The complex is comprised of two lateral elements and a central element connected by perpendicular transverse filaments (TFs). A 3D model based on actual morphological data of the SC is missing. Here, we applied electron tomography (ET) and manual feature extraction to generate a quantitative 3D model of the murine SC. We quantified the length (90 nm) and width (2 nm) of the TFs. Interestingly, the 80 TFs/µm are distributed asymmetrically in the central region of the SC challenging available models of SC organization. Furthermore, our detailed 3D topological analysis does not support a bilayered organization of the central region as proposed earlier. Overall, our quantitative analysis is relevant to understand the functions and dynamics of the SC and provides the basis for analyzing multiprotein complexes in their morphological context using ET.