Rescued Secretion of Misfolded Mutant Proinsulin.

In pancreatic beta cells, the insulin precursor proinsulin is folded in the endoplasmic reticulum (ER), forming three critical intramolecular disulfide bonds. After homo-dimerizing, native proinsulin exits the ER en route to secretory vesicles, where it forms hexamers, is endoproteolytically cleaved to mature insulin, and is stored until it is secreted in response to elevated blood glucose. In Mutant Ins-gene induced Diabetes of Youth (MIDY), misfolded mutant proinsulin is retained in the ER and acts in a dominant-negative manner to impair maturation of wild-type (wt) proinsulin, leading to decreased insulin release and eventual ER stress-induced beta cell death. Using cell culture and mouse models, I have investigated two potential mechanisms to improve secretion of misfolded mutant proinsulin. First, I found that intermolecular interactions between proinsulin molecules impact strongly on the fate of those molecules. Misfolded mutant proinsulin molecules dimerize with and impair secretion of co- expressed wt molecules. Interestingly, the opposite is also true; wt proinsulin molecules also stabilize and enhance secretion of mutant molecules. Thus, there is a dynamic bidirectional interaction between dimerization partners, which we hope to exploit pharmacologically to improve clearance of misfolded proteins from the ER and alleviate ER stress-induced cell death. In the second half of my project, I investigated how manipulating the oxidative environment of the ER may impact proinsulin secretion and beta cell health in cells expressing mutant proinsulin. ER Oxidoreductin-1 (Ero1), the best-studied ER oxidant, contributes to oxidative folding of secretory proteins by coupling generation of de novo disulfide bonds with reduction of molecular oxygen. Due to its generation of hydrogen peroxide as a byproduct, Ero1 hyperactivity has been speculated to contribute to cell death in stressed beta cells. Surprisingly, I found the opposite to be true. Overexpression of Ero1 rescued secretion of wt proinsulin in the presence of mutant proinsulin. Furthermore, Ero1 directly rescued a subset of MIDY mutant proinsulins by improving their oxidative folding, resulting in a decrease in mutant proinsulin-induced ER stress response. These findings improve our understanding of proinsulin maturation in beta cells, and may contribute to novel therapeutic approaches in this and other secretory protein conformational diseases.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111355/1/jordwrig_1.pd

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

The Dynamics of Folded Nascent Chains Studied With NMR Spectroscopy

Efficient protein folding is vital for biological function. In the cell, this process can begin during biosynthesis, as proteins gradually emerge from the ribosome exit tunnel. Such progressive, co-translational folding of partially synthesised fragments can increase folding efficiency, by reducing the risk of misfolding between adjacent domains. However, the high effective concentration of the ribosome surface itself may potentially also modulate co-translational folding, by forming interactions with the nascent chain which stabilise folded or unfolded states. Solution-state NMR spectroscopy is a uniquely powerful tool to study the conformation and dynamics of the nascent chain beyond the ribosome exit tunnel and probe such effects. The method has previously been applied to characterise the progressive emergence and folding of the FLN5 filamin domain and quantify interactions between the ribosome surface and the unfolded state. However, interactions with folded nascent chains have not so far been studied. As folded states have very different spectroscopic properties to previously studied unfolded states, in this work we sought to develop a toolkit of NMR methods optimised for the analysis of dynamics folded nascent chains. Firstly, we describe the optimisation using mass-spectroscopy of methyl label incorporation into perdeuterated, translationally-arrested ribosome-nascent chain complexes. Secondly, we report the development and application of sensitivityoptimised measurements of relaxation and cross-correlated relaxation in methyl spin systems, which reveal a reduction in the mobility or rotational diffusion of folded FLN5 RNCs. Finally, we combine these approaches with a protein engineering strategy to explore the determinants of folded nascent chain dynamics during translation. We find that mobility increases as translation progresses, but always remains significantly lower than the isolated protein regardless of linker length or composition. We attribute this observation to interactions with the ribosome surface, which we show to be at least partly electrostatic in nature

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

Cotranslational Pulling Forces Alter Outcomes of Protein Synthesis

As nascent proteins are synthesized by the ribosome, interactions between the nascent protein and its environment can create pulling forces that are transmitted to the ribosome's catalytic center. These forces can affect the rate and outcomes of translation. We use atomistic and coarse-grained simulation to characterize the origins of pulling forces, the propagation of force to catalytic center of the ribosome, and the effects of force on synthetic outcomes. We uncover a novel form of pulling force-mediated regulation in which the forces generated by the integration of a transmembrane helix induce frameshifting in a viral polyprotein. Computational force measurements of hundreds of mutant viral sequences in combination with deep mutational scanning experiments reveal the structural and sequence-level features that enable this powerful regulatory mechanism. Force measurements are also used to provide a molecular picture for complex pulling force experiments on multispanning membrane proteins. In particular, we identify signatures of cotranslational helix packing interactions and the translocation of surface helices. To understand how forces are propagated through the nascent protein in the ribosomal exit tunnel, we ran and analyzed hundreds of microseconds of atomistic molecular dynamics with an applied pulling force on the nascent protein. The simulations reveal how the secondary structure of nascent proteins and their interactions with the ribosome control force propagation. The inhibition of force transduction by nascent protein-ribosome interactions explains how amino acids tens of angstroms away from the catalytic center of the ribosome can still influence the force-induced restart of stalled ribosomes.</p

Analysis of the role of N-terminal acetylation of newly synthesized proteins in Saccharomyces cerevisiae

N-terminal acetylation is a conserved co-translational protein modification that is highly abundant among eukaryotes. In Saccharomyces cerevisiae, at least five enzymes with distinct substrate specificities (N-terminal acetyl transferase Nat A to E) act to acetylate 50–70% of the yeast proteins. Despite being one of the most common protein modifications, its biological significance remains largely ambiguous. I set out to study the role of N-terminal acetylation in yeast cells by employing quantitative proteomics and ribosome profiling for analysis of the consequences of failure of N-terminal acetylation in strains lacking specific N-terminal acetyl transferases. My results revealed a multi-faceted stress response in natB deletion mutant that modulates protein quality control machinery, protein biogenesis capacity, and energy regeneration pathways in order to establish protein homeostasis. Systematic analysis of proteome stability in the natB deletion mutant suggests no global effect of the loss of N-terminal acetylation on the turnover of NatB substrates, but an increase in the level of global protein aggregation. SILAC-based mass spectrometry analysis of aggregated proteins isolated from the natB deletion mutant shows no significant enrichment of NatB substrates, indicating that protein aggregation in the natB deletion mutant cannot be solely explained as a direct consequence of the loss of N-terminal acetylation. In contrast, these protein aggregates show strong enrichment for components of specific biological pathways, in particular of the translation apparatus, suggesting an underlying selective sequestration mechanism. Consistently, quantitative proteomics revealed that, on average, approximately 40% of each of the quantified ribosomal proteins is sequestered into protein aggregates in the natB deletion mutant. Moreover, the aggregated proteins showed significantly higher interaction between each other and overlapped with aggregated proteins generated upon environmental stress, suggesting a common mode of sequestration of proteins into aggregates. Interestingly, the aggregated proteins in the natB deletion mutant strongly overlap with those identified upon deletion of the genes encoding the ribosome-associated Hsp70 chaperone Ssb. In addition, deletion of SSB in the natB deletion mutant leads to synthetic growth defects. Moreover, isolation of radiolabeled protein aggregates after 5 min 35S pulse labeling showed that a fraction of the newly synthesized proteins is readily sequestered into aggregates. These findings together suggest a new link between N-terminal acetylation by NatB and co-translational protein folding activity by Ssb. Parallel analysis of natA deletion mutant revealed similar protein aggregation patterns, suggesting a general role of N-terminal acetylation in the maintenance of proteome integrity

Structural and spectroscopic studies on the porin-cytochrome complex from Shewanella oneidensis MR-1

The"outer"membrane,"hetero!trimeric"multi!heme"cytochrome"complex"MtrCAB,"enables"the" process"of"dissimilatory"metal"reduction"(DMR)"in"Shewanella(oneidensis." "" The"properties"of" the"decaheme"protein"MtrA"as"well"as"a" truncated"version"of" this"protein" were" investigated" using" analytical" ultracentrifugation" (AUC)," small" angle" X!ray" scattering" (SAXS)" and" spectropotentiometric" techniques." MtrA" and" a" truncated"N!terminal" MtrA" construct"containing" the" five"N!terminal"hemes"(MtrA"N)"were"both"observed" to"be"prolate" and"highly"extended" along"one" axis."Through" aligning"MtrA"N"with"MtrA," the"N" terminus" of" the"MtrA"structure"was"identified.""Redox"titration"experiments"were"performed"on"MtrA"as" well" as" N" and"C" terminal" truncations."From" these" titrations" three"distinct" groups" of" hemes" were" identified;" a" high," a" middle" potential" and" a" low" potential" group" of" hemes." The" five"N" terminal"hemes"contained"the"high"and"middle"potential"groups"of"hemes;"whereas"the"five"C" terminal"hemes"contained"the"middle"and"low"potential"groups"of"hemes." " The"MtrCAB" complex"was" inserted" into" liposomes" containing" either"methyl" viologen," small" tetra" heme" cytochrome" (STC)" or" cytochrome" c." Using" these" proteoliposomes" the" rate" of" electron"transfer"across"MtrCAB"was"investigated."MtrCAB"was"seen"to"enable"the"reduction" and"oxidation"of"methyl"viologen"and"STC,"but"only"the"reduction"of"cytochrome"c.""STC"was" reduced" during" the" experiments," implying" an" electron" storage" role" in" the" periplasm" during"respiration." "Finally,"the" structure" of" MtrCAB" was" investigated" through" small" angle" neutron" scattering" (SANS)."Structures"of"MtrCAB"were"highly"elongated"with"a"globular"head" region"and"a" tail" region."Through"density"matching"buffers,"scattering"produced"from"detergents"was"removed" and"a"prediction"of"the"relative"location"of"MtrB"within"the"model"made."The"model"produced" was" found" to" be" long" enough" to" span" the" periplasm" enabling" direct" contact" of"MtrA" with" proteins"on"the"inner"membrane."

Reprogramming of Sterol Biosynthesis in Chinese Hamster Ovary Cells for Enhanced Recombinant Protein Production

The biopharmaceutical industry is a multi-billion dollar global market with projections that within the next 5 to 10 years, up to 50% of all drugs in development will be biopharmaceuticals. The majority of these drugs are recombinantly produced protein based therapeutics with the demand for such new therapeutic products continuing to increase, broadening the range of medical conditions that they are being used to treat. Chinese hamster ovary (CHO) cells are the preferred and most commonly used system for large scale production of recombinant glycoproteins for therapeutic use due to their ability to perform complex human-like post-translational modifications required for biological activity. CHO cells also display robust growth, high productivity and stability, attributes that have enabled them to become widely used. Whilst there have been large increases in the yield and quality of recombinant protein that can be obtained from mammalian expression systems over the years, particularly CHO cells, many recombinant proteins remain difficult to express in CHO cells or any other system. Many of these proteins are highly complex, highly glycosylated, large and unstable proteins. As such, there remains a need to develop CHO cell systems that are able to produce such difficult to express proteins rapidly in higher yield and quality. The endoplasmic reticulum (ER) is a key compartment in the secretory pathway of mammalian cells and during the production of recombinant proteins in CHO cells this has reportedly been a potential bottleneck and site of perceived cellular stress as a result of the load imposed on the cell by the recombinant protein. This study has investigated two sterol reductases; Transmembrane 7 Superfamily member 2 (TM7SF2) and 7-dehydrocholesterol reductase (DHCR7). ER resident proteins involved in the biosynthesis of cholesterol that have been reported to influence endoplasmic reticulum (ER) and nuclear envelope (NE) expansion when over-expressed in mammalian cell lines. The work described here therefore set out to determine whether overexpression of these sterol reductases could enhance the secretory capacity of CHO cells by expansion of the ER and hence lead to increased yields of difficult to express recombinant proteins compared to those cells where these were not overexpressed. The TM7SF2 and DHCR7 genes were cloned into a mammalian expression vector with a hygromycin selection marker to allow for the selection of stable cell lines where the genes had been integrated into the genome. Stable CHO cells overexpressing TM7SF2 and DHCR7 were generated to assess the impact on cell growth and stable and transient recombinant protein expression, monitored using western blot techniques and microscopy analysis. Control cell lines over-expressing GFP (but not the sterol reductases) and the pcDNA3.1Hygro (empty) vector were also developed. Stable expression levels of the sterol reductases were determined in heterogeneous pools and then used for limited dilution cloning to isolate clonal cell lines with differing sterol reductase expression which were further investigated. Growth profile assays were undertaken to monitor any toxicity effect of over-expression of TM7SF2 and DHCR7 on CHO cells. Transient expression studies of the recombinant bio therapeutic protein etanercept, a 150 kDa biomolecule and erythropoietin (EPO), a 36 kDa recombinant protein, were carried out to investigate the production capacity of lipid engineered CHO cells with reference to controls. Immunocytolocalisation studies were also undertaken to investigate pools and clones of lipid targets predicted to be ER-localized or associated. A YFP tag was present on the N-terminal of TM7SF2 and DHCR7 molecules and was used to monitor expression by fluorescence in stable CHO cells. The data from these experiments show that both TM7SF2 and DHCR7 engineered CHO cells had improved cell growth and culture viability over the course of 10-day experimental study compared to the control cells. Western blot studies showed that both TM7SF2 and DHCR7 could be stably over-expressed in CHO cells and the amount of expression varied among the generated clones. Further, the high expressers of TM7SF2 and DHCR7 were identified and investigated for EPO and etanercept production and expression levels of these recombinant biomolecules were observed to be proportional to the levels of TM7SF2 and DHCR7 expressions in CHO cells. More importantly, TM7SF2 and DHCR7 engineered cells showed increased expression levels of these recombinant products when compared with pcDNA3.1Hygro controls as determined by western blot analysis of the amount of secreted recombinant target protein in the cell culture supernatant. Confocal images also showed clones expressed different levels of YFP signal which related to expression levels observed when western blot techniques were used. In summary, the data presented here shows that the manipulation of cellular circuits such as cholesterol biosynthesis has the potential to enhance cellular growth and recombinant protein yield. By designing new hosts and cellular circuits to reprogramme the CHO cell ER there is the potential of expanding the secretory capacity and/or subsequent secretory vesicle system