Hijacked then lost in translation:the plight of the recombinant host cell in membrane protein structural biology projects

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells

ER import of small human presecretory proteins: components and mechanisms

Protein transport into the mammalian endoplasmic reticulum (ER) used to be seen as strictly cotranslational, that is temporarily and mechanistically coupled to protein synthesis. In the course of the last decades, however, several classes of precursors of soluble and membrane proteins were found to be post-translationally imported into the ER, without any involvement of the ribosome. The first such class to be identified were the small presecretory proteins; tail-anchored membrane proteins followed next. In both classes, the inherent address tag is released from the translating ribosome before the initiation of ER import, as part of the fully synthesized precursor. In small presecretory proteins, the information for ER targeting and -translocation via the polypeptide-conducting Sec61-channel is encoded by a classical N-terminal signal peptide, which is released from the ribsosome before targeting due to the small size of the full-length precursor. Here, we discuss the current state of research on targeting and translocation of small presecretory proteins into the mammalian ER. In closing, we present a unifying hypothesis for ER protein translocation in terms of an energy diagram for Sec61-channel gating

Cotranslational folding of proteins on the ribosome.

Many proteins in the cell fold cotranslationally within the restricted space of the polypeptide exit tunnel or at the surface of the ribosome. A growing body of evidence suggests that the ribosome can alter the folding trajectory in many different ways. In this review, we summarize the recent examples of how translation affects folding of single-domain, multiple-domain and oligomeric proteins. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. Finally, the assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding

The Molecular Basis For Post And Co-Translational N-Terminal Acetylation

N-terminal acetylation is among the most common protein modifications in eukaryotic cells, where up to 90% of proteins carry this mark. Over the last decade, significant progress has been made in elucidating the function of N-terminal acetylation for a number of diverse systems. The enzymes responsible for this modification are the N-terminal acetyltransferases (NATs). The NATs are a group of conserved enzymes in eukaryotes, which can be broadly classified into two groups. The major NATs, NatA-NatC, are promiscuous and responsible for acetylating the majority of N-termini in the cell, and have hundreds to thousands of protein substrates. Recently, it has been suggested that they also may target lysine residues. These NATs largely act co-translationally. They interact with the ribosome near the exit tunnel and acetylate the nascent protein chain as it is being translated. The other NATs, NatD-NatF, are more specialized in their substrate specificity and localization patterns. There is a large amount of structural information on the molecular basis of NAT activity. These studies have revealed how the NATs are specific for N-termini, and how binding partners affect their activity. However, the structural characteristics of the specialized NATs remain unknown, the full substrate scope of NATs remains to be determined, and how the NATs interact with the ribosome is largely unexplored, despite the importance of this modification in a co-translational context. Therefore, to address these gaps in knowledge, we determined the crystal structures of NatD and NatF with their substrate peptides bound, and by carrying out biochemical and enzymological assays, we revealed the molecular basis for their unique specificity and localization. We also showed that there is no difference in lysine acetylation of substrate proteins with or without NatA, suggesting that reported substrates are acetylated chemically rather than enzymatically. Finally, we determined regions on the NatA complex that promote interaction with the ribosome. This dissertation provides a framework for understanding the molecular basis of the specialized NATs, and how NATs interact with the ribosome for co-translational protein acetylation. These studies have implications in developing inhibitors for the specialized NATs, and in understanding co-translational events in general

Mechanisms of ER Protein Import

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of human cells, which amounts to about 30% of the proteome. Most of these proteins fulfill their functions either in the membrane or lumen of the ER plus the nuclear envelope, in one of the organelles of the pathways for endo- and exocytosis (ERGIC, Golgi apparatus, endosome, lysosome, and trafficking vesicles), or at the cell surface as plasma membrane or secreted proteins. An increasing number of membrane proteins destined to lipid droplets, peroxisomes or mitochondria are first targeted to and inserted into the ER membrane prior to their integration into budding lipid droplets or peroxisomes or prior to their delivery to mitochondria via the ER-SURF pathway. ER protein import involves two stages, ER targeting, which guarantees membrane specificity, and the insertion of nascent membrane proteins into or translocation of soluble precursor polypeptides across the ER membrane. In most cases, both processes depend on amino-terminal signal peptides or transmembrane helices, which serve as signal peptide equivalents. However, the targeting reaction can also involve the ER targeting of specific mRNAs or ribosome–nascent chain complexes. Both processes may occur co- or post-translationally and are facilitated by various sophisticated machineries, which reside in the cytosol and the ER membrane, respectively. Except for resident ER and mitochondrial membrane proteins, the mature proteins are delivered to their functional locations by vesicular transport

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease

Profiling interactions of proximal nascent chains reveals a general co-translational mechanism of protein complex assembly

The association of proteins into functional oligomeric complexes is crucial for nearly all cellular processes. Despite rapid progress in characterizing the structure of native assemblies, the underlying mechanisms that guide faithful complex formation in the crowded cellular environment are understood only superficially. To secure efficient complex biogenesis and limit the exposure of aggregation-prone intermediates, many proteins assemble co-translationally, via interaction of a fully synthetized and a nascent protein subunit (co-post assembly). Here, we explore the prevalence and the mechanistic principles of a putative co-translational assembly mechanism, which involves the direct interaction of nascent subunits emerging from proximal ribosomes (co-co assembly). To obtain direct evidence of this putative assembly mode, we apply a newly developed method based on Ribosome Profiling, named Disome Selective Profiling (DiSP), which allows to monitor the conversion of single ribosomes to nascent chain connected ribosome pairs across the proteome with high resolution. We use this approach to analyse co-co assembly in two human cell lines and demonstrate that it constitutes a general mechanism inside cells that is employed by hundreds of high confidence and thousands of low confidence candidates, comprising 11 to 32% of all complex subunits. Analysing the features of the co-co assembly proteome, we reveal that this mechanism guides formation of mostly homomeric complexes and typically relies on interaction of N-terminal nascent chain segments. We further identify five dimerization domains mediating the majority of co-co interactions, which are either partially or completely exposed at the onset of nascent chain dimerization, implying different folding and assembly mechanisms. The detectable fraction of each candidate’s nascent chains that co-co assemble is in median 40% and in some cases exceeds 90%, suggesting that this co-translational assembly path may be employed as the main route for complex formation. To gain deeper insights into the mechanistic basis of co-co assembly, we took a series of experimental approaches that distinguish between interactions of nascent chains emerging from the same or different polysomes (termed assembly in cis and in trans, respectively). These experiments could not support a model of assembly in trans. Conversely, we find indications supporting a cis assembly model for nuclear lamin C, one of our high confidence candidates. This mechanism provides a simple explanation for the remarkable specificity of lamin homodimer formation in vivo, where splice variants with largely overlapping sequences do not mix. We propose that assembly in cis more generally secures specific homomer formation of isoforms and structurally-related proteins which are highly prone to promiscuous interactions inside cells. In conclusion, this study provides a global annotation of nascent chain interactions across the human proteome and elucidates the basic principles of this widespread assembly pathway. Our findings raise a number of fundamental questions concerning the mechanisms ensuring high-fidelity protein biogenesis, including the implications of co-co assembly on polysome structure, the possible consequences of co-co assembly failure, the inter-dependence with co-translational folding and the synchronization and coordination with translation kinetics

Systems Biology of Protein Secretion in Human Cells: Multi-omics Analysis and Modeling of the Protein Secretion Process in Human Cells and its Application.

Since the emergence of modern biotechnology, the production of recombinant pharmaceutical proteins has been an expanding field with high demand from industry. Pharmaceutical proteins have constituted the majority of top-selling drugs in the pharma industry during recent years. Many of these proteins require post-translational modifications and are therefore produced using mammalian cells such as Chinese Hamster Ovary cells. Despite frequent improvements in developing efficient cell factories for producing recombinant proteins, the natural complexity of the protein secretion process still poses serious challenges for the production of some proteins at the desired quantity and accepted quality. These challenges have been intensified by the growing demands of the pharma industry to produce novel products with greater structural complexity,\ua0\ua0as well as increasing expectations from regulatory authorities in the form of new quality control criteria to guarantee product safety.This thesis focuses on different aspects of the protein secretion process, including its engineering for cell factory development and analysis in diseases associated with its deregulation. A major part of this thesis involved the use of HEK293 cells as a human model cell-line for investigating the protein secretion process by generating different types of omics data and developing a computational model of the human protein secretion pathway. We compared the transcriptomic profile of cell lines producing erythropoietin (EPO; as a model secretory protein) at different rates to identify key genes that potentially contributed to higher rates of protein secretion. Moreover, by performing a transcriptomic comparison of cells producing green fluorescent protein (GFP; as a model non-secretory protein) with EPO producers, we captured differences that specifically relate to secretory protein production. We sought to further investigate the factors contributing to increased recombinant protein production by analyzing additional omic layers such as proteomics and metabolomics in cells that exhibited different rates of EPO production. Moreover, we developed a toolbox (HumanSec) to extend the reference human genome-scale metabolic model (Human1) to encompass protein-specific reactions for each secretory protein detected in our proteomics dataset. By generating cell-line specific protein secretion models and constraining the models using metabolomics data, we could predict the top host cell proteins (HCPs) that compete with EPO for metabolic and energetic resources.\ua0Finally,\ua0based on the detected patterns of changes in our multi-omics investigations combined with a protein secretion sensitivity analysis using the metabolic model, we identified a list of genes and pathways that potentially play a key role in recombinant protein production and could serve as promising candidates for targeted cell factory design.In another part of the thesis, we studied the link between the expression profiles of genes involved in the protein secretory pathway (PSP) and various hallmarks of cancer. By\ua0implementing a dual approach involving differential expression analysis and eight different machine learning algorithms, we investigated the expression changes in secretory pathway components across different cancer types to identify PSP genes whose expression was associated with tumor characteristics. We demonstrated that a combined machine learning and differential expression approach have a complementary nature and could highlight key PSP components relevant to features of tumor pathophysiology that may constitute potential therapeutic targets

Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution

BACKGROUND: The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis. RESULTS: I elucidate nuclear and mitotic coevolution, explaining the origins of dicer and small centromeric RNAs for positionally controlling centromeric heterochromatin, and how 27 major features of the cell nucleus evolved in four logical stages, making both mechanisms and selective advantages explicit: two initial stages (origin of 30 nm chromatin fibres, enabling DNA compaction; and firmer attachment of endomembranes to heterochromatin) protected DNA and nascent RNA from shearing by novel molecular motors mediating vesicle transport, division, and cytoplasmic motility. Then octagonal nuclear pore complexes (NPCs) arguably evolved from COPII coated vesicle proteins trapped in clumps by Ran GTPase-mediated cisternal fusion that generated the fenestrated nuclear envelope, preventing lethal complete cisternal fusion, and allowing passive protein and RNA exchange. Finally, plugging NPC lumens by an FG-nucleoporin meshwork and adopting karyopherins for nucleocytoplasmic exchange conferred compartmentation advantages. These successive changes took place in naked growing cells, probably as indirect consequences of the origin of phagotrophy. The first eukaryote had 1-2 cilia and also walled resting cysts; I outline how encystation may have promoted the origin of meiotic sex. I also explain why many alternative ideas are inadequate. CONCLUSION: Nuclear pore complexes are evolutionary chimaeras of endomembrane- and mitosis-related chromatin-associated proteins. The keys to understanding eukaryogenesis are a proper phylogenetic context and understanding organelle coevolution: how innovations in one cell component caused repercussions on others. REVIEWERS: This article was reviewed by Anthony Poole, Gáspár Jékely and Eugene Koonin