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While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions. [Display omitted] • NPR mRNA contains a CUG and an AUG TIS, each producing a distinct proteoform • Alternative TIS usage in NPR is regulated by RNA structures and neuronal activity • AUG initiation shortens the signal sequence and converts NPR to a secreted factor • Altering NPR proteoform ratio impacts AMPA receptors in PV+ neurons and behaviors Unlike most mRNAs that encode one protein, Lee et al. discover that several neuronal synaptic organizer mRNAs encode more than one protein product (proteoform) by using multiple translation initiation sites. Alternative translation initiation remodels the N-terminal signal sequence, diversifies proteoform localization and functions, and impacts animal behaviors. [ABSTRACT FROM AUTHOR]

The signal sequence played a crucial role in the efficacy of mRNA vaccines against virus pandemic by influencing antigen translation. However, limited research had been conducted to compare and analyze the specific mechanisms involved. In this study, a novel approach was introduced by substituting the signal sequence of the mRNA antigen to enhance its immune response. Computational simulations demonstrated that various signal peptides differed in their binding capacities with the signal recognition particle (SRP) 54 M subunit, which positively correlated with antigen translation efficiency. Our data revealed that the signal sequences of tPA and IL-6-modified receptor binding domain (RBD) mRNA vaccines sequentially led to higher antigen expression and elicited more robust humoral and cellular immune protection against the SARS-CoV-2 compared to the original signal sequence. By highlighting the importance of the signal sequence, this research provided a foundational and safe approach for ongoing modifications in signal sequence-antigen design, aiming to optimize the efficacy of mRNA vaccines. [ABSTRACT FROM AUTHOR]

Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time. [ABSTRACT FROM AUTHOR]

• The intergenic regions of mitogenomes may have functional and/or regulatory roles that have yet to be identified. • Here, we bioinformatically investigated and characterised the motif of "WHWGHTW" in nd4L - (X) - trnT gene cluster in mitogenome across 1889 species from Bilateria. • This motif can be functional especially in mitochondrial transcription because of its reverse complementarity to a previously described DmTTF binding sequence (Roberti et al., 2006, 2003). • The motif is remarkable because of its possession of G and T nucleotides in the fourth and sixth positions, and, the bias on T and G nucleotides instead of C and A in the degenerated positions. The animal mitogenomes which undergone a reductive evolution has an obvious loss of coding capacity compared to their known closest relatives, but it has not yet been fully investigated why and how the intergenic regions do not encode protein and have no known functions, are stably maintained, replicated, and transmitted by the genome. These relatively small intergenic regions may not be under neutral evolution and they may have functional and/or regulatory roles that have yet to be identified. Here, the distribution pattern, sequence content and location of a novel sequence motif of 'WWWGHTW' were bioinformatically investigated and characterised by constructing a sampling mitogenome dataset of 1889 species from 14 phyla representing the clade of Bilateria. This motif is reverse complementary of the previously described DmTTF binding sequence and found in the nd4L - (X) -trnT gene cluster. This cluster commonly exhibits a strand displacement region and an intergenic region among the bilaterian superphylums, particularly in Ecdysozoa. This motif may be accepted as a substrate providing binding sites for the specific interaction with transcription factors because of (i) its reverse complementarity of previously described DmTTF binding sequence, and (ii) the possession of G and T nucleotides in the fourth and sixth positions, (iii) the bias on T and G nucleotides instead of C and A in the degenerated positions. This suggestion is also supported by the presence of a strand displacement region in the nd4L - (X) -trnT gene cluster, particularly in Ecdysozoa consisting of the most rearranged mitogenomes among the bilaterian superphylums. [ABSTRACT FROM AUTHOR]

Background: Disease caused by the capsular group B meningococcus (MenB) is the leading cause of infectious death in UK infants. A novel adenovirus-based vaccine encoding the MenB factor H binding protein (fHbp) with an N-terminal dual signal sequence induces high titres of protective antibody after a single dose in mice. A panel of N-terminal signal sequence variants were created to assess the contribution of components of this sequence to transgene expression kinetics of the encoded antigen from mammalian cells and the resultant effect on immunogenicity of fHbp. Results: The full-length signal sequence (FL SS) resulted in superior early antigen expression compared with the panel of variants, as measured by flow cytometry and confocal imaging, and supported higher bactericidal antibody levels against the expressed antigen in mouse sera < 6 weeks post-immunisation than the licensed four component MenB vaccine. The FL SS also significantly increased antigen-specific T cell responses against other adenovirus-encoded bacterial antigens in mice. Conclusions: These findings demonstrate that the FL SS enhances immunogenicity of the encoded antigen, supporting its inclusion in other viral vectored bacterial antigen transgenes. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum represents the gateway to the secretory pathway. Here, proteins destined for secretion, as well as soluble and membrane proteins that reside in the endomembrane system and plasma membrane, are triaged from proteins that will remain in the cytosol or be targeted to other cellular organelles. This process requires the faithful recognition of specific targeting signals and subsequent delivery mechanisms to then target them to the translocases present at the ER membrane, which can either translocate them into the ER lumen or insert them into the lipid bilayer. This review focuses on the current understanding of the first step in this process representing the targeting phase. Targeting is typically mediated by cleavable N-terminal hydrophobic signal sequences or internal membrane anchor sequences; these can either be captured co-translationally at the ribosome or recognised post-translationally and then delivered to the ER translocases. Location and features of the targeting sequence dictate which of several overlapping targeting pathway substrates will be used. Mutations in the targeting machinery or targeting signals can be linked to diseases. [ABSTRACT FROM AUTHOR]

In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel (PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex. SYNOPSIS: The overall architecture of the eukaryotic SecY/Sec61 channel for post‐translational protein translocation, and how it recognizes N‐terminal signal sequences in transported proteins, remain unclear. Here, structural analyses reveal the molecular architecture of the engaged yeast Sec complex, which binds the signal sequence in a unique open conformation. Cryo‐EM structures of the heptameric Sec complex reveal the conformational transition from the partially open apo‐state to the fully open signal sequence‐bound state.The signal sequence binds the lateral gate of the Sec61 complex, which is stabilized by the transmembrane helices of the Sec62 subunit.The X‐ray structure of the N‐terminal cytoplasmic domain of Sec62 reveals a novel fold that is coordinated by the phosphorylated C‐terminal peptide of the Sec63 subunit. [ABSTRACT FROM AUTHOR]

• pre-Ost, an engineered signal sequence for improved protein secretion in yeast. • Bioreactor-scale evaluation of pre-Ost's full potential in Pichia pastoris. • The evaluation was carried out with three different model proteins. • The new signal sequence can easily outperform the pre-α-MF in Pichia pastoris. • The new signal sequence enhances cell performance in bioreactor cultures. Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved. [ABSTRACT FROM AUTHOR]

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display. [ABSTRACT FROM AUTHOR]

[Display omitted] • Sequences from 560,000 proteins modeled in DeepMind's AlphaFold2 database have been submitted to the signal/transit peptide prediction algorithms TargetP 2.0 and SignalP 6.0. • When applied to proteins known experimentally to contain signal/transit peptides, AlphaFold2 almost always builds the signal/transit peptide in a conformation that is disengaged from the body of the protein. • The presence of close contacts between a predicted signal/transit peptide and the body of the protein in the predicted AlphaFold2 structure appears to be an excellent diagnostic for identifying true positive and false positive signal peptide predictions. • Analysis of the AlphaFold2 structures strongly suggests that both TargetP 2.0 and SignalP 6.0 are highly accurate methods: the majority of false positive predictions are ones that were predicted with low confidence, while higher confidence predictions tend to be true positives. Many proteins contain cleavable signal or transit peptides that direct them to their final subcellular locations. Such peptides are usually predicted from sequence alone using methods such as TargetP 2.0 and SignalP 6.0. While these methods are usually very accurate, we show here that an analysis of a protein's AlphaFold2-predicted structure can often be used to identify false positive predictions. We start by showing that when given a protein's full-length sequence, AlphaFold2 builds experimentally annotated signal and transit peptides in orientations that point away from the main body of the protein. This indicates that AlphaFold2 correctly identifies that a signal is not destined to be part of the mature protein's structure and suggests, as a corollary, that predicted signals that AlphaFold2 folds with high confidence into the main body of the protein are likely to be false positives. To explore this idea, we analyzed predicted signal peptides in 48 proteomes made available in DeepMind's AlphaFold2 database (https://alphafold.ebi.ac.uk). Applying TargetP 2.0 and SignalP 6.0 to the 561,562 proteins in the database results in 95,236 being predicted to contain a cleavable signal or transit peptide. In 95.1% of these cases, the AlphaFold2 structure of the full-length protein is fully consistent with the prediction of TargetP 2.0 or SignalP 6.0. In the remaining 4.9% of cases where the AlphaFold2 structure does not appear consistent with the prediction, the signal is often only predicted with low confidence. The potential false positives identified here may be useful for training even more accurate signal prediction methods. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, biogenesis of proteins destined to the secretory pathway begins from the cytosol. Nascent chains are either co-translationally or post-translationally targeted to the endoplasmic reticulum (ER) and translocated across the membrane through the Sec61 complex. For the post-translational translocation, the Sec62/Sec63 complex is additionally required. Sec63, however, is also shown to mediate co-translational translocation of a subset of proteins, the types and characteristics of proteins that Sec63 mediates in translocation still await to be defined. To overview the types of proteins that require Sec63 for the ER translocation, we prepared Sec63 mutant lacking the first 39 residues (Sec63_ΔN39) in yeast and assessed initial translocation efficiencies of diverse types of precursors in the sec63_ΔN39 strain by a 5 min metabolic labeling. By employing Blue-Native gel electrophoresis (BN-PAGE), stability of the SEC complex (Sec61 plus Sec62/Sec63 complexes) isolated from cells carrying the Sec63_ΔN39 mutant was examined. Among the various translocation precursors tested, we found that proper sorting of single- and double-pass membrane proteins was severely impaired in addition to post-translational translocation precursor in the sec63_ΔN39 mutant strain. Stability of the SEC complex was compromised upon deletion of the N-terminal 39 residues. The N-terminus of Sec63 is important for stability of the SEC complex and Sec63 is required for proper sorting of membrane proteins in vivo. Sec63 is essential on insertion of membrane proteins. • The N-terminal deletion of Sec63 impairs membrane insertion of proteins. • The N-terminus of Sec63 is important for stability of the SEC complex. • Sec63 is required for proper insertion and topogenesis of membrane proteins. [ABSTRACT FROM AUTHOR]

Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression. Unlabelled Image • Mutations in secretory proteins are associated with many human diseases. • Disease-causing mutations affecting interaction with SRP trigger mRNA degradation. • SRP has a dual function in protein secretion and in protection of the mRNAs from degradation. [ABSTRACT FROM AUTHOR]

Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished—the signal, the stop-transfer sequence, and the re-integration sequence—which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones. [ABSTRACT FROM AUTHOR]

Abstract The N-terminal signal sequence (SS) on proteins targeted to the endoplasmic reticulum (ER) is surprisingly diverse in hydrophobicity, in the number of preceding N-terminal residues (N-length), and in charged residues flanking the sequence. However, it remains unclear how these sequences despite their heterogeneity bind to the same site and open the Sec61 translocon. We assessed varying features of SSs and their efficiencies in initiating translocation across the ER by using 5-min radiolabeling in yeast. We found that while hydrophobic SSs with a short N-length efficiently initiated translocation in Sec62 mutant, Sec63 mutant and Sec72 deletion strains, most SSs showed varying degrees of translocation defect in these strains. In particular, Sec71 was required for internal hydrophobic SSs to efficiently initiate translocation. These results suggest that different combinations of Sec62, Sec63, Sec71 and Sec72 dynamically associate with the Sec61 translocon for the optimal binding of incoming SSs of broad characteristics and the initiation of protein translocation in vivo. Highlights • Most signal sequences require Sec62/Sec63 complex to initiate protein translocation. • Sec62/Sec63 complex subunits dynamically sort signal sequences of varying features. • Sec71 is necessary for internal signal sequences. [ABSTRACT FROM AUTHOR]

Introduction Cellular and humoral immune responses are both involved in protection against Plasmodium infections. The only malaria vaccine available, RTS,S, primarily induces short-lived antibodies and targets only a pre-erythrocytic stage antigen. Inclusion of erythrocytic stage targets and enhancing cellular immunogenicity are likely necessary for developing an effective second-generation malaria vaccine. Adenovirus vectors have been used to improve the immunogenicity of protein-based vaccines. However, the clinical assessment of adenoviral-vectored malaria vaccines candidates has shown the induction of robust Plasmodium -specific CD8+ but not CD4+ T cells. Signal peptides (SP) have been used to enhance the immunogenicity of DNA vaccines, but have not been tested in viral vector vaccine platforms. Objectives The objective of this study was to determine if the addition of the SP derived from the murine IgGκ light chain within a recombinant adenovirus vector encoding a multistage P. vivax vaccine candidate could improve the CD4+ T cell response. Methods In this proof-of-concept study, we immunized CB6F1/J mice with either the recombinant simian adenovirus 36 vector containing the SP (SP-SAd36) upstream from a transgene encoding a chimeric P. vivax multistage protein or the same SAd36 vector without the SP. Mice were subsequently boosted twice with the corresponding recombinant proteins emulsified in Montanide ISA 51 VG. Immunogenicity was assessed by measurement of antibody quantity and quality, and cytokine production by T cells after the final immunization. Results The SP-SAd36 immunization regimen induced significantly higher antibody avidity against the chimeric P. vivax proteins tested and higher frequencies of IFN-γ and IL-2 CD4+ and CD8+ secreting T cells, when compared to the unmodified SAd36 vector. Conclusions The addition of the murine IgGκ signal peptide significantly enhances the immunogenicity of a SAd36 vectored P. vivax multi-stage vaccine candidate in mice. The potential of this approach to improve upon existing viral vector vaccine platforms warrants further investigation. [ABSTRACT FROM AUTHOR]

The B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labelling and electron paramagnetic resonance spectroscopy to wild-type (wt) EAL from Salmonella typhimurium, by using the sulphydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as βC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. Electron paramagnetic resonance (EPR) spectra of 4MT-labelled wt EAL are collected over 200-265 K in frozen, polycrystalline water-only, and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow-reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labelling of a single site (βC37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC. [ABSTRACT FROM AUTHOR]

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing posttranslational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n = 10 or 20]. The yields of the (SP)n-fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. [ABSTRACT FROM AUTHOR]

Recombinant Pasterurella multocida lipoprotein E (PlpE) has been shown to protect against fowl cholera. This study aimed to determine if the signal sequence may contribute to the antigenicity and protective efficacy of recombinant PlpE. A small antigenic domain of PlpE (termed truncated PlpE, tPlpE) was constructed with (SP-tPlpE) or without (tPlpE) the signal sequence and evaluated in vitro and in vivo. In vitro, the HEK-Bule hTLR2 Cells were used to evaluate the activation of NF-kB in the test associated with the stimulation of the SP-tPlpE and tPlpE proteins. When chickens were immunized, compared to the tPlpE vaccine group, the SP-tPlpE group showed higher antibody levels and enhanced CD4+ T cell response. In a challenge test, the SP-tPlpE group showed a survival rate of 87.5% (n = 8), compared to 25% for the tPlpE group. It is confirmed that the inclusion of the native signal sequence enhanced protective efficacy against fowl cholera and may act as a vaccine adjuvant. The short SP-tPlpE construct is amenable to further vaccine engineering and has potential to be developed as a fowl cholera vaccine. [ABSTRACT FROM AUTHOR]

Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control. [ABSTRACT FROM AUTHOR]

We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting. [ABSTRACT FROM AUTHOR]

The low density lipoprotein receptor (LDLR) is a transmembrane protein that plays a key role in cholesterol metabolism. It contains 860 amino acids including a 21 amino acid long signal sequence, which directs the protein into the endoplasmic reticulum. Mutations in the LDLR gene lead to cholesterol accumulation in the plasma and results in familial hypercholesterolemia (FH). Knowledge of the impact of a mutation on the LDLR protein structure and function is very important for the diagnosis and management of FH. Unfortunately, for a large proportion of mutations this information is still missing. In this study, we focused on the LDLR signal sequence and carried out functional and in silico analyses of two sequence changes, p.(Gly20Arg) and p.(Leu15Pro), localized in this part of the LDLR. Our results revealed that the p.(Gly20Arg) change, previously described as disease causing, has no detrimental effect on protein expression or LDL particle binding. In silico analysis supports this observation, showing that both the wt and p.(Gly20Arg) signal sequences adopt an expected α-helix structure. In contrast, the mutation p.(Leu15Pro) is not associated with functional protein expression and exhibits a structure with disrupted a α-helical arrangement in the signal sequence, which most likely affects protein folding in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Many membrane proteins possessing hydrophobic transmembrane (TM) segments are cotranslationally integrated into the endoplasmic reticulum (ER) membrane. Various peroxisomal and mitochondrial membrane proteins escape the ER-targeting mechanism and are targeted to their destinations. Here, we discovered a short segment in the 70-kDa peroxisomal membrane protein (PMP70) that suppresses ER targeting. The first TM segment has an intrinsic signal function that targets the nascent chain to the ER. The ER targeting was suppressed by a short N-terminal sequence of nine residues that is 80 residues upstream of the TM segment. Among the nine residues, Ser5 is indispensable. The short segment also suppressed the signal peptide function of an authentic secretory protein. This function of the short segment was suppressed by the recombinant motif-GST fusion protein. The 50-kDa and 20-kDa proteins were crosslinked with the motif. The PMP70 molecule with the Ser5Ala point mutation predominantly localized to the ER. We propose the concept of an ER-targeting suppressor that suppresses the ER-targeting mechanism via a binding factor. [ABSTRACT FROM AUTHOR]

[Display omitted] • Human SRP targets secretory proteins to ER and protects their mRNAs. • SRP54 depletion globally downregulates secretory and membrane protein mRNAs. • SRP54 knockdown upregulates specific chaperone network and ubiquitination. • Loss of SRP54 leads to ribosomal stress and change in expression of RPS27 and RPS27L. • Data demonstrate complex nature of SRP function and dramatic consequences of defects. 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. [ABSTRACT FROM AUTHOR]

The biosynthesis of thousands of proteins requires targeting a signal sequence or transmembrane segment (TM) to the endoplasmic reticulum (ER). These hydrophobic ɑ helices must localize to the appropriate cellular membrane and integrate in the correct topology to maintain a high-fidelity proteome. Here, we show that the P5A-ATPase ATP13A1 prevents the accumulation of mislocalized and misoriented proteins, which are eliminated by different ER-associated degradation (ERAD) pathways in mammalian cells. Without ATP13A1, mitochondrial tail-anchored proteins mislocalize to the ER through the ER membrane protein complex and are cleaved by signal peptide peptidase for ERAD. ATP13A1 also facilitates the topogenesis of a subset of proteins with an N-terminal TM or signal sequence that should insert into the ER membrane with a cytosolic N terminus. Without ATP13A1, such proteins accumulate in the wrong orientation and are targeted for ERAD by distinct ubiquitin ligases. Thus, ATP13A1 prevents ERAD of diverse proteins capable of proper folding. [Display omitted] • ATP13A1 dislocates mitochondrial TA proteins that mislocalize to the ER via the EMC • SPP cleaves mislocalized mitochondrial TA proteins for ERAD • ATP13A1 mediates signal sequence and type II transmembrane protein topogenesis • Distinct ubiquitin ligases target misoriented proteins for ERAD McKenna et al. show that corrective quality control by the P5A-ATPase ATP13A1 at the ER prevents wasteful ER-associated degradation of mislocalized and misoriented proteins capable of folding properly. [ABSTRACT FROM AUTHOR]

Ostensive signals preceding referential cues are crucial in communication-based human knowledge acquisition processes. Since dogs are sensitive to both human ostensive and referential signals, here we investigate whether they also take into account the order of these signals and, in an object-choice task, respond to human pointing more readily when it is preceded by an ostensive cue indicating communicative intent. Adult pet dogs ( n = 75) of different breeds were presented with different sequences of a three-step human action. In the relevant sequence (RS) condition, subjects were presented with an ostensive attention getter (verbal addressing and eye contact), followed by referential pointing at one of two identical targets and then a non-ostensive attention getter (clapping of hands). In the irrelevant sequence (IS) condition, the order of attention getters was swapped. We found that dogs chose the target indicated by pointing more frequently in the RS as compared to the IS condition. While dogs selected randomly between the target locations in the IS condition, they performed significantly better than chance in the RS condition. Based on a further control experiment ( n = 22), it seems that this effect is not driven by the aversive or irrelevant nature of the non-ostensive cue. This suggests that dogs are sensitive to the order of signal sequences, and the exploitation of human referential pointing depends on the behaviour pattern in which the informing cue is embedded. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 (DE3) and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space. [ABSTRACT FROM AUTHOR]

Periplasmic localization of recombinant proteins offers advantages over cytoplasmic protein expression. In this study signal sequence of amicyanin, which is encoded by the mauC gene of Paracoccus denitrificans , was used to express the light chain variable domain of the human κIO8/O18 germline antibody in the periplasm of Escherichia coli . The expressed protein was purified in good yield (70 mg/L of culture) in one step from the periplasmic fraction by affinity chromatography using an engineered hexahistidine tag. Circular dichroism spectroscopy was used to determine if the secondary and tertiary structures of the protein and its thermal stability corresponded to those of the native folded protein. The expressed and purified protein was indeed properly folded and exhibited a reasonable thermal transition temperature of 53 °C. These results indicate that the amicyanin signal sequence may be particularly useful for prokaryotic expression of proteins which are prone to mis-folding, aggregation or formation of inclusion bodies, all of which were circumvented in this study. [ABSTRACT FROM AUTHOR]

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP- RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP-FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery. [ABSTRACT FROM AUTHOR]

The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP–signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the N ε -(5-azido-2 nitrobenzoyl)-Lys-tRNA amb amber suppressor. A homogeneous population of SRP–ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP–signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP–signal sequence affinity and targeting to the translocon. [ABSTRACT FROM AUTHOR]

The chloroplast plays critical roles in lipid metabolism of microalgae, thus it is recognized as an attractive target of metabolic engineering to enhance biofuel production. It has been well known that recombinant protein expression in microalgal chloroplasts needs specific signal sequence which governs the transition manner of nuclear-encoded polypeptides within the subcellular compartments. However certain microalgae, including diatoms, have complex membrane systems surrounding the chloroplast, and thus chloroplast-targeting protein expression with the signal sequence has rarely been demonstrated except for a few model non-oleaginous diatoms. In this study, we performed recombinant green fluorescence protein (GFP) expression and transportation into the chloroplast of the oleaginous marine diatom, Fistulifera solaris JPCC DA0580. The signal sequence of ATP synthetase gamma subunit, which was predicted to localize in the chloroplast according to a bioinformatics analysis pipeline, was employed as a key factor of this technique. As a result, specific localization of GFP in the chloroplast was observed. It would be useful to engineer the lipid synthesis pathways existing in the chloroplast. Furthermore, intensive gathering of GFP in the rod-like structure was also detected, which has not been observed in model diatom studies. As comparing with electron microscopic observation, the structure was estimated to be a pyrenoid. [ABSTRACT FROM AUTHOR]

Background Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. Results One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. Conclusions With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion. [ABSTRACT FROM AUTHOR]

Highlight: [•] Signal sequence for green fluorescent protein production in Aspergillus oryzae was selected. [•] Signal sequence from Taka-amylase A promoted higher secretional protein production. [•] Fusion of signal sequence and N28 fragment improved secretory protein production. [•] Repeated signal sequence also induced secretory protein production. [Copyright &y& Elsevier]

Highlights: [•] Leptin receptor (LepR) contains less efficient signal sequence. [•] Enhanced signal sequence efficiency promotes LepR expression. [•] Enhanced signal sequence efficiency potentiates leptin signaling. [•] Therapeutic enhancement of LepR translocation for leptin resistance. [ABSTRACT FROM AUTHOR]

Accurate folding, assembly, localization, and maturation of newly synthesized proteins are essential to all cells and require high fidelity in the protein biogenesis machineries that mediate these processes. Here, we review our current understanding of how high fidelity is achieved in one of these processes, the cotranslational targeting of nascent membrane and secretory proteins by the signal recognition particle (SRP). Recent biochemical, biophysical, and structural studies have elucidated how the correct substrates drive a series of elaborate conformational rearrangements in the SRP and SRP receptor GTPases; these rearrangements provide effective fidelity checkpoints to reject incorrect substrates and enhance the fidelity of this essential cellular pathway. The mechanisms used by SRP to ensure fidelity share important conceptual analogies with those used by cellular machineries involved in DNA replication, transcription, and translation, and these mechanisms likely represent general principles for other complex cellular pathways. [ABSTRACT FROM AUTHOR]

Highlights: [•] Expression of full-length KIAA1199 is essential for HA depolymerizing activity. [•] The cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199. [•] The deletion of the N-terminal portion results in altered localization of KIAA1199. [•] The N-terminal 30 amino acids of KIAA1199 are indeed the cleavable signal sequence. [Copyright &y& Elsevier]

Highlights: [•] C-terminal extensions to Sdh3 and Shh3 result in SRP-dependent ER mis-targeting. [•] Mitochondrial membrane proteins evade SRP recognition by having short C-terminal tails after strongly hydrophobic segments. [•] Eluding SRP recognition may be one way that hydrophobic membrane proteins ensure proper targeting to mitochondria. [ABSTRACT FROM AUTHOR]

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity. [ABSTRACT FROM PUBLISHER]

Background: Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication. Results: To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. Conclusions: C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6) – three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30 – obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6. The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments [Copyright &y& Elsevier]

Abstract: Regulating the entry of secretory and membrane proteins into the ER is shown to be an important physiological process, in terms of controlling quantity, localization and therefore function of target proteins. However, few small molecule modulators are available to pharmacologically regulate translocation of a specific protein. Here, we identified a small molecule that specifically inhibits pathogenic PrP biosynthesis using a highly sensitive and reproducible assay based on the fluorogenic substrate reporter PrP. This molecule specifically destabilized the signal peptide of PrP, leading to cotranslational rerouting of a significant amount of nascent PrP to proteasome-dependent degradation pathway in the cytosol. This study suggests that regulating differential translocation efficiency attributable to sequence diversity in signal peptides of disease-causing secretory and membrane proteins using cell-permeable small molecules may be a potential therapeutic approach for diseases associated with impaired protein biogenesis. [Copyright &y& Elsevier]

The signal recognition particle (SRP) and its receptor compose a uni-versally conserved and essential cellular machinery that couples the syn-thesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investi-gations of this targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how die SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP-SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed. [ABSTRACT FROM AUTHOR]

Highlights: [•] A taste-modifier protein named miraculin is secreted outside the plasma membrane. [•] This secretion is dependent on a signal peptide conserved in Kunitz-type STIs. [•] Transgenic seedlings of miraculin were cultured in liquid medium. [•] Miraculin was present in the supernatant after cellulase treatment. [•] Miraculin may have lost trypsin inhibitory activity during its evolution. [ABSTRACT FROM AUTHOR]

Abstract: Although clusterin (CLU) was originally identified as a secreted glycoprotein that plays cytoprotective role, several intracellular CLU variants have been recently identified in the diverse pathological conditions. The mechanistic basis of these variants is now believed to be alternative splicing and retrotranslocation. Here, we uncovered, an unglycosylated and signal sequence-unprocessed, CLU variant in the cytosol. This variant proved to be a product that cotranslationally rerouted to the cytosol during translocation. Cytosolic CLU was prone to aggregation at peri-nuclear region of cells and induced cell death. Signal sequence is shown to be an important determinant for cytosolic CLU generation and aggregation. These results provide not only a new mechanistic insight into the cytosolic CLU generation but also an idea for therapeutic mislocalization of CLU as a strategy for cancer treatment. [Copyright &y& Elsevier]

Abstract: Most mitochondrial proteins are encoded in the nucleus. They are synthesized as precursor forms in the cytosol and must be imported into mitochondria with the help of different protein translocases. Distinct import signals within precursors direct each protein to the mitochondrial surface and subsequently onto specific transport routes to its final destination within these organelles. In this review we highlight common principles of mitochondrial protein import and address different mechanisms of protein integration into mitochondrial membranes. Over the last years it has become clear that mitochondrial protein translocases are not independently operating units, but in fact closely cooperate with each other. We discuss recent studies that indicate how the pathways for mitochondrial protein biogenesis are embedded into a functional network of various other physiological processes, such as energy metabolism, signal transduction, and maintenance of mitochondrial morphology. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. [Copyright &y& Elsevier]

Abstract: Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors. [Copyright &y& Elsevier]

This article proposes a method of moments technique for estimating the sparsity of signals in a random sample. This involves estimating the largest eigenvalue of a large Hermitian trigonometric matrix under mild conditions. As illustration, the method is applied to two well-known problems. The first focuses on the sparsity of a large covariance matrix and the second investigates the sparsity of a sequence of signals observed with stationary, weakly dependent noise. Simulation shows that the proposed estimators can have significantly smaller mean absolute errors than their main competitors. [ABSTRACT FROM PUBLISHER]

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF. [ABSTRACT FROM AUTHOR]

Abstract: Aggregation is an important concern for therapeutic antibodies, since it can lead to reduced bioactivity and increase the risk of immunogenicity. In our analysis of immunoglobulin G (IgG) molecules of identical amino acid sequence but produced either in mammalian cells (HEK293) or in the yeast Pichia pastoris (PP), dramatic differences in their aggregation susceptibilities were encountered. The antibodies produced in Pichia were much more resistant to aggregation under many conditions, a phenomenon found to be mainly caused by two factors. First, the mannose-rich glycan of the IgG from Pichia, while slightly thermally destabilizing the IgG, strongly inhibited its aggregation susceptibility, compared to the complex mammalian glycan. Second, on the Pichia-produced IgGs, amino acids belonging to the α-factor pre-pro sequence were left at the N-termini of both chains. These additional residues proved to considerably increase the temperature of the onset of aggregation and reduced the aggregate formation after extended incubation at elevated temperatures. The attachment of these residues to IgGs produced in cell culture confirmed their beneficial effect on the aggregation resistance. Secretion of IgGs with native N-termini in the yeast system became possible after systematic engineering of the precursor proteins and the processing site. Taken together, the present results will be useful for the successful production of full-length IgGs in Pichia, give indications on how to engineer aggregation-resistant IgGs and shed new light on potential biophysical effects of tag sequences in general. [Copyright &y& Elsevier]

Polypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum (ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We propose that positive charges are independent translocation regulators that are more active than previously believed. [ABSTRACT FROM AUTHOR]

Cloning and expression of any heterologous gene product always impart metabolic load on the recombinant host for utilization of its metabolites that results in variations in cellular physiology. An understanding of such stress is important for robust process design and scale-up. In the present communication, an attempt has been made to explain the metabolic load imposed on the recombinant strain of Escherichia coli BL21(DE3) owing to over-expression of amyE gene, using different expression vectors of varying size. Current studies show that metabolic quotient or specific oxygen uptake rate (SOUR) of different recombinant hosts increases with an increase in vector size, showing correlation between vector size and cellular metabolic load. Further increase in SOUR on protein expression shows even higher metabolic load for protein expression than plasmid maintenance. Increase in bacterial size and decrease in specific growth rate on transformation and recombinant protein expression are cellular responses to additional metabolic load. [ABSTRACT FROM AUTHOR]