Signal Peptide Cleavage Research Articles

A Gain-of-Function Mutation in EPO Gene Causes Familial Erythrocytosis

Mutations in the erythropoietin-receptor gene (EPOR) are well known causes of inherited forms of primary erythrocytosis that is characterized by low serum erythropoietin (Epo) levels. Secondary erythrocytosis with elevated Epo serum levels is mainly caused by mutations in genes involved in oxygen sensing pathway. Epo is the primary regulator of erythropoiesis, but mutations in the EPO gene have so far not been described as a cause or erythrocytosis. Here we studied a Norwegian family with autosomal dominant erythrocytosis and elevated Epo serum levels.We performed genome-wide linkage analysis using SNP arrays on 16 informative family members including 10 affected individuals and identified a co-segregating region on chromosome 7q22.1 with a LOD score of 3.3. Targeted sequencing of all 215 genes within the co-segregating region revealed only a heterozygous single base deletion in exon 2 of EPO as the sole candidate gene mutation. ThisΔG deletion creates a frame-shift that truncates the Epo signal peptide and generates a novel peptide, terminating after additional 51 amino acids. Thus, contrary to the expectation based on the erythrocytosis phenotype, the EPO ΔG deletion was predicted to be a loss-of-function mutation.Since we had no access to patient's kidney or liver tissues where Epo is produced, we used the CRISPR technique to introduce the ΔG mutation into the EPO gene into Hep3B cells, a human hepatoma cell line that expresses EPO . We obtained two Hep3B single-cell derived clones (ΔG1 and ΔG4) that were homozygous for the EPO ΔG mutation and measured Epo protein in the culture supernatants. We found that these supernatants contained 8-10 times more Epo than parental Hep3B cells or controls, as determined by ELISA (Figure 1A), and were also capable of stimulating the growth of an Epo-dependent cell line (BaF3- EpoR), demonstrating biological activity (Figure 1B). Thus, the ΔG deletion introduced into the EPO gene locus in Hep3B cells behaved as a gain-of-function mutation.To decipher the mechanism of how the ΔG mutation was able to produce the excess of Epo protein, we searched for alternative EPO mRNAs by 5'-RACE. In addition to the expected transcripts originating from the physiologic promoter (P1) upstream of exon 1, we discovered two types of alternative mRNAs that initiate in intron 1 of EPO. Both initiatied from a putative alternative promoter (P2) in intron 1 that contains two GATA motifs. In addition to Hep3B ΔG clones, P2 transcripts were also detected in parental Hep3B cells and also in mRNA from normal kidney and liver.To determine which transcripts are capable of producing Epo, we transfected HEK293 cells with cDNAs representing P1 or P2 transcripts with or without the ΔG mutation. Supernatants of cells transfected with P2 ΔG transcripts contained more Epo than cells transfected with the wildtype P1 cDNA. These supernatants also stimulated the growth of Epo dependent BaF3- EpoR cells and supported erythroid colony formation of progenitors from human peripheral blood, demonstrating that P2 ΔG transcripts produce biologically active Epo protein.Analysis of the P2 mRNA sequences shows that the normally non-coding P2 transcripts through the ΔG single base deletion connect to the Epo coding sequence creating a functional signal peptide with a novel N-terminus, but retaining the physiologic Epo signal peptide cleavage site. These P2 ΔG transcripts are more abundant in liver and produce an excess of biologically active Epo by escaping the oxygen sensing regulation. Our data demonstrate for the first time mutation in EPO as a cause of familial erythrocytosis and provide an explanation of how the ΔG results in a gain-of-function mutation (Figure 1C).Figure 1 legend: A) Production of Epo protein by He3B cells carrying the ΔG mutation. Supernatants of parental Hep3B or CRISPR-modified Hep3B clones were collected and Epo protein was measured by Epo ELISA. The levels of Epo are given as a mean ± SD (n=3), p < 0.001 = *** as determined by one-way ANOVA. B) Biological activity of Epo produced by parental Hep3B or CRISPR-modified Hep3B cells measured by a proliferation assay with BaF3-EpoR cell line. BaF3-EpoR cells were grown in serial dilutions of supernatants and cell numbers were assessed by Cell Titer-Glo Assay, (n=3). Serial dilutions of 2.5 IU/ml of recombinant human Epo were used for reference. C) Model and proposed mechanism for EPO overproduction by a single nucleotide deletion in exon 2 of the EPO gene. [Display omitted] DisclosuresWaage:Celgene, Takeda: Honoraria. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau.

Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1.

ABSTRACTPestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2.IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

Mining the 99 Lives Cat Genome Sequencing Consortium database implicates genes and variants for the Ticked locus in domestic cats (Feliscatus).

SummaryTabby patterns of fur coats are defining characteristics in wild and domestic felids. Historically, three autosomal alleles at one locus (Tabby): Abyssinian (Ta; a.k.a. ticked), mackerel (Tm; a.k.a. striped) and blotched (tb; a.k.a. classic, blotched) were thought to control these patterns in domestic cats and their breeds. Currently, at least three loci influence cat tabby markings, two of which are designated Tabby and Ticked. The Tabby locus is laeverin (LVRN) and affects the mackerel and blotched patterns. The unidentified gene for the Ticked locus on cat chromosome B1 was suggested to control the presence or absence of the ticked pattern (Tabby – Abyssinian (Ta; a.k.a. ticked). The cat reference genome (Cinnamon, the Abyssinian) has the ticked phenotype and the variant dataset and coat phenotypes from the 99 Lives Cat Genome Consortium (195 cats) were used to identify candidate genes and variants associated with the Ticked locus. Two strategies were used to find the Ticked allele(s), one considered Cinnamon with the reference allele or heterozygous (Strategy A) and the other considered Cinnamon as having the variant allele or heterozygous (Strategy B). For Strategy A, two variants in Dickkopf Wnt Signaling Pathway Inhibitor 4 (DKK4), a p.Cys63Tyr (B1:41621481, c.188G>A) and a less common p.Ala18Val (B1:42620835, c.53C>T) variant are suggested as two alleles influencing the Ticked phenotype. Bioinformatic and molecular modeling analysis suggests that these changes disrupt a key disulfide bond in the Dkk4 cysteine‐rich domain 1 or Dkk4 signal peptide cleavage respectively. All coding variants were excluded as Ticked alleles using Strategy B.

A Partial Phenotype of adFNDI Related to the Signal Peptide c.55G&gt;A Variant of the AVP Gene

The autosomal dominant familial form of neurohypophyseal diabetes insipidus (adFNDI) is a rare inherited endocrine disorder characterized by hypotonic polyuria, severe thirst and polydipsia, which results from a deficient neurosecretion of the antidiuretic hormone, also known as arginine vasopressin (AVP). To date, adFNDI has been linked to more than 70 different heterozygous point mutations of the 2.5 kb AVP gene, encoding the composite precursor protein of AVP. A minority of disease-causing mutations, such as the common c.55G&gt;A variant, are predicted to affect amino acid residues close to the signal peptide (SP) cleavage site, and result in abnormal post-translational processing and intracellular trafficking of AVP precursors exerting neurotoxic activity on vasopressinergic magnocellular neurons. Generally, SP variants cause a gradual decline in the neurohypophyseal secretion of AVP in small children, although a wide variability in clinical onset and severity of manifestations has been reported. For the first time, we describe a kindred from Calabria (Southern Italy) with adFNDI and document a partial clinical phenotype in one female young adult member of the family. Methods: A young adult woman was subjected to clinical, neuroradiological and genetic assessments for a mild, adolescent-onset, polyuric state at our Endocrinology Unit. Her family medical history revealed an early-onset (&lt;12 years of age) occurrence of polyuria and polydipsia, which was successfully managed with high doses of oral desmopressin, and a typical adFNDI inheritance pattern that was seen over three generations. Results: In the index patient, the extensive hypertonic dehydration during fluid deprivation test elicited a prompt elevation of urine osmolality and diuresis contraction, indicative of a partial adFNDI phenotype. Diagnosis was confirmed by concordant hormonal tests and magnetic resonance imaging (MRI) evidence of a reduced hyperintense signal of the neurohypophysis, which was regarded as compatible with the depletion of the vasopressinergic magnocellular neurons. Direct DNA sequencing and restriction enzyme cleavage analysis revealed that a heterozygous c.55G&gt;A transition, predicting a p.Ala19Thr replacement in the C-terminal region of SP, was the cause of adFNDI in the investigated kindred. Conclusions: The identification of the genetic cause of aFNDI in this Calabrian kindred provides further information and confirms the wide variability of disease onset and severity of manifestations related to SP variants of the AVP gene, supporting the need for genetic testing in all patients with familial occurrence of polyuria, regardless of their clinical and radiological phenotype. Even though sexual differences in the antidiuretic responses are documented, it is unclear whether female gender would attenuate clinical disease progression in the presence of a pathogenic c.55G&gt;A mutation.

English

Post-translational modifications (PTMs) regulate different complex mechanisms of cell and affect cell growth, stress, evolution of living organisms and adaptations due to environment. The purpose of the present research is to investigate the genetic diversity and PTMs of protease (Carboxypeptidase A1) n Sordaria fimicola. They perform a variety of functions ranging from housekeeping: e.g., protein maturation, signal peptide cleavage, signal transduction, intracellular protein turnover, immune response, apoptosis, and reproduction. S. fimicola is a microscopic filamentous fungus, has been preferably used in this study because of its easy growing pattern on Potato Dextrose Agar (PDA) and a short life cycle of 7 to 12 days. The genomic DNA of six of the strains S. fimicola was used to amplify the carboxypeptidases A1 gene (CpA1), the product size was 940 bp. The multiple sequence alignment of the nucleotide sequences of six strains of S. fimicola with Neurospora crassa (as a reference strain) was studied. The numbers of polymorphic sites in six strains of S. fimicola with respect to N. crassa were six. Posttranslational modifications were depicted by using bioinformatics tools i.e., YinOYang1.2, NetPhos 3.1 and NetNES 1.1 Server to calculate O-glycosylation, phosphorylation sites, and nuclear export signals respectively. The study has predicted 19 phosphorylation sites on serine residues for protease Carboxypeptidase A1 in S1 strains of S. fimicola while 15 phosphorylation sites on serine in N7 strain and 17 serine phosphorylation modifications were predicted in N. Crassa. The results of this research will be helpful for further in vitro investigations of this industrially important enzyme under study. © 2021 Friends Science Publishers

Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments.

Bacteriophages that infect Gram-negative bacteria as source of signal-arrest-release motif lysins

Treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria is challenging, a potential solution for which is the use of bacteriophage-derived lytic enzymes. However, the exogenous action of bacteriophage lysins against Gram-negative bacteria is hindered due to the presence of an impermeable outer membrane in these bacteria. Nevertheless, recent research has demonstrated that some lysins are capable of permeating the outer membrane of Gram-negative bacteria with the help of signal peptides. In the present study, we investigated the genomes of 309 bacteriophages that infect Gram-negative pathogens of clinical interest in order to determine the evolutionary markers of signal peptide-containing lysins. Complete genomes displayed 265 putative lysins, of which 17 (6.41%) contained signal-arrest-release motifs and 41 (15.47%) contained cleavable signal peptides. There was no apparent relationship between host specificity and lysin diversity. Nevertheless, the evolution of lysin genes might not be independent of the rest of the bacteriophage genome once pan-genome clustering and lysin diversity appear to be correlated. In addition, signal peptide- and signal-arrest-release-containing lysins were monophyletically distributed in the protein cladogram, suggesting that the natural selection of holin-independent lysins is divergent. Our study screened 58 (21.89%) out of 265 potential candidates for in vitro experimentation against MDR bacteria.

Miniature Seed6, encoding an endoplasmic reticulum signal peptidase, is critical in seed development.

Endoplasmic reticulum (ER) type I signal peptidases (ER SPases I) are vital proteases that cleave signal peptides from secreted proteins. However, the specific function of ER SPase I in plants has not been genetically characterized, and the substrate is largely unknown. Here, we report the identification of a maize (Zea mays) miniature seed6 (mn6) mutant. The loss-of-function mn6 mutant exhibited severely reduced endosperm size. Map-based cloning and molecular characterization indicated that Mn6 is an S26-family ER SPase I, with Gly102 (box E) in Mn6 critical for protein function during processing. Mass spectrometric and immunoprecipitation analyses revealed that Mn6 is predominantly involved in processing carbohydrate synthesis-related proteins, including the cell wall invertase miniature seed1 (Mn1), which is specifically expressed in the basal endosperm transfer layer. RNA and protein expression levels of Mn1 were both significantly downregulated in the mn6 mutant. Due to the significant reduction in cell wall invertase activity in the transfer cell layer, mutation of Mn6 caused dramatic defects in endosperm development. These results suggest that proper maturation of Mn1 by Mn6 may be a crucial step for proper seed filling and maize development.

SecY-mediated quality control prevents the translocation of non-gated porins

OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.

Yeast-based reporter assay system for identifying the requirements of intramembrane proteolysis by signal peptide peptidase of Arabidopsis thaliana.

Signal peptide peptidase (SPP) is an aspartic protease with two active sites, YD and GXGD, in the transmembrane domain. SPP cleaves signal peptides, and the released fragments play key roles in the immune system, embryo development and protein turnover in cells. Despite SPP having an important function, a general system to identify the requirements of intramembrane proteolysis by SPP has not been developed because proteolysis occurs in the membrane. In this study, we first established a reporter assay system in yeast to verify the cleavage activity of the Arabidopsis thaliana SPP (AtSPP). Next, we screened candidate substrates of AtSPP from A. thaliana pollen and roots. In the pollen, 13 signal peptides with 'pollen' and 'cell wall' as gene ontology terms were selected. In the roots, mutants overexpressing AtSPP were constructed, and gene expression changes were compared with the wild-type. Nine signal peptides expressed in the roots were selected. Then we used the candidate substrates in our reporter assay system to determine the requirements for proteolysis by AtSPP. Fifteen of 22 signal peptides were cleaved by AtSPP. The absence of the positively charged amino acids, His and Lys on the C terminus of the signal sequence, was observed in cleaved substrates. Moreover, mutation of a helix breaker-to-Leu substitution in the intramembrane region in substrates prevented cleavage by AtSPP. These results indicated that substrates of AtSPP required the helix breaker structure to be cleaved.

Direct detection of intact Klebsiella pneumoniae carbapenemase variants from cell lysates: Identification, characterization and clinical implications

IntroductionCarbapenemase-producing organisms (CPOs) are a growing threat to human health. Among the enzymes conferring antibiotic resistance produced by these organisms, Klebsiella pneumoniae carbapenemase (KPC) is considered to be a growing global health threat. Reliable and specific detection of this antibiotic resistance-causing enzyme is critical both for effective therapy and to mitigate further spread. ObjectivesThe objective of this study is to develop an intact protein mass spectrometry-based method for detection and differentiation of clinically-relevant KPC variants directly from bacterial cell lysates. The method should be specific for any variant expressed in multiple bacterial species, limit false positive results and be rapid in nature to directly influence clinical outcomes. MethodsLysates obtained directly from bacterial colonies were used for intact protein detection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bottom-up and top-down proteomic methods were used to characterize the KPC protein targets of interest. Comparisons between KPC-producing and KPC-non-producing isolates from a wide variety of species were also performed. ResultsCharacterization of the mature KPC protein revealed an unexpected signal peptide cleavage site preceding an AXA signal peptide motif, modifying the molecular weight (MW) of the mature protein. Taking the additional AXA residues into account allowed for direct detection of the intact protein using top-down proteomic methods. Further validation was performed by transforming a KPC-harboring plasmid into a negative control strain, followed by MS detection of the KPC variant from the transformed cell line. Application of this approach to clearly identify clinically-relevant variants among several species is presented for KPC-2, KPC-3, KPC-4 and KPC-5. ConclusionDirect detection of these enzymes contributes to the understanding of occurrence and spread of these antibiotic-resistant organisms. The ability to detect intact KPC variants via a simple LC-MS/MS approach could have a direct and positive impact on clinical therapy, by providing both direction for epidemiological tracking and appropriate therapy.

Rational design and expression of a recombinant plant rhabdovirus glycoprotein for production of immunoreactive murine anti-sera

Lettuce necrotic yellows virus (LNYV) is a plant rhabdovirus which has a type-1 transmembrane glycoprotein. Here, we describe the generation of murine anti-sera to the glycoprotein. Rational design, expression, and purification of recombinant glycoprotein, termed rLGe, was undertaken using SignalP4.1 and camSol servers to predict signal peptide cleavage and protein solubility. In order to successfully obtain expression in mammalian cells, LNYV glycoprotein native signal peptide was substituted with that of Rabies virus glycoprotein. In addition, rather than expression of the entire molecule, rLGe consisted of the LNYV glycoprotein ectodomain fused to two affinity tags to minimize the risk of protein aggregation, while facilitating detection and purification. rLGe was transiently expressed in mammalian cell culture, purified using affinity column chromatography, and used to immunize mice. Harvested anti-sera were immunoreactive and specific to the naturally occurring glycoprotein as confirmed by western blotting of plant leaf tissue infected with LNYV.

Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum.

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.

2108-P: Complex Posttranslational Processing of Glucagon Prohormone in Human Pancreatic Islets

An array of signaling peptides and peptide hormones in pancreatic islets results from posttranslational processing of precursor proteins by well-characterized endopeptidases. While the encoded peptide complement can be predicted in silico from the prohormone sequence, actual peptide products often are more diverse due to additional chemical or enzymatic modifications of mature predicted peptides. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for comprehensive molecular characterization, we compiled an experimentally deduced database of endogenous peptides in human pancreatic islets and revealed an unexpected complex processing of several prohormones including proglucagon. Live pancreatic islets from several donors were provided by the Human Pancreas Analysis Program, University of Pennsylvania. Total of 2034 chemically unique peptide sequences were identified and matched to 477 proteins in the human protein database. Among interesting observations, we find differential cleavage of the signal peptide on the glucagon precursor protein, NP_002045.1, and unusual further processing of its encoded mature peptides. Alternate signal peptide cleavage sites are confirmed by detection of both classical GRPP, R[21]-D[50], and its novel variant S[18]-D[50]. An amidated IP-2 D[131]-L[142] and an isoform of MPGF D[131]-D[178], missing the GLP-1 sequence were detected. Previously unreported N-terminus acetylation of glucagon, GLP-1, GLP-1 (7-36), and oxyntomodulin was detected. Novel peptides potentially resulted from variable processing of mature glucagon, GRPP, and oxyntomodulin at internal convention cleavage sites were characterized. New chemically unique peptides originating from proglucagon and documented in this study indicate a more complex posttranslational processing of proglucagon in human islets than previously thought. The finding expands opportunity for mechanistic studies on pathology, progression, and treatments of diabetes. Disclosure E.V. Romanova: None. S. Rubakhin: None. J.V. Sweedler: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-18-VSN-19 to J.V.S.)

Identification of Ala2Thr mutation in insulin gene from a Chinese MODY10 family.

More than 80% of maturity-onset diabetes of the young (MODY) in Chinese is genetically unexplained. To investigate whether the insulin gene (INS) mutation is responsible for some Chinese MODY, we screened INS mutations causing MODY10 in MODY pedigrees and explored the potential pathogenic mechanisms. INS mutations were screened in 56 MODY familial probands. Structure-function characterization and clinical profiling of identified INS mutations were conducted. An INS mutation, at the position 2 alanine-to-threonine substitution (A2T), was identified and co-segregated with hyperglycemia in a MODY pedigree. The A2T mutation converted an α-helix into a β-sheet at the N-terminal of the signal peptide (SP) of preproinsulin. The A2T mutation did not affect preproinsulin translocation across endoplasmic reticulum (ER) membrane, but impaired its SP cleavage within the ER. In INS-1 cells transfected with an A2T mutant, glucose-stimulated insulin secretion (GSIS) was significantly decreased, while BiP luciferase activities were significantly increased compared to that of wild type (WT). We identified an INS-A2T mutation cosegregating with diabetes in a Chinese MODY pedigree. This mutation severely impaired SP cleavage and thus blocked the formation of proinsulin, resulting in enhanced ER stress, which may be responsible for decreased insulin secretion and subsequently, the onset of MODY10.

Cleavage of FNDC5 and insights into its maturation process

FNDC5 corresponds to an irisin precursor that increases with exercise. Studies suggest that irisin mediates beneficial effects in adipose tissues, skeletal muscle, bone, and brain. However, the cleavage and maturation processes of FNDC5 have not been clearly identified. This study aimed to show that the signal peptide and transmembrane domain of FNDC5 were associated with the secretion of its ectodomain. Localization studies identified the signal peptide that was responsible for endoplasmic reticulum targeting activity of nascent FNDC5 and showed that the FNDC5 ectodomain corresponding to irisin could be transported across the membrane by a transmembrane domain. Analysis of cleavage constructs revealed that the ectodomain of FNDC5 could be cleaved from its signal peptide and transmembrane attachment. Genetic ablation of the signal peptide cleavage site blocked N-glycosylation of FNDC5. Identification of the FNDC5 maturation process should facilitate our understanding of irisin secretion.

Effect of changes at the conserved + 3 position of mature archaellins on in vitro cleavage by the pre-archaellin peptidase FlaK of Methanococcus maripaludis.

Archaea swim using archaella that are domain-specific rotary type IV pilus-like appendages. The structural components of the archaellum filament are archaellins, initially made as preproteins with type IV pilin-like signal peptides which are removed by signal peptidases that are homologues of prepilin peptidases that remove signal peptides from type IV pilins. N-terminal sequences of archaellins, including the signal peptide cleavage site, are conserved and various positions have been previously shown to be critical for signal peptide removal. Archaellins have an absolute conservation of glycine at the + 3 position from the signal peptide cleavage site. To investigate its role in signal peptide cleavage, I used archaellin variants in which the + 3 glycine was mutated to all other possibilities in in vitro cleavage reactions. Cleavage was observed with ten different amino acids at the + 3 position, indicating that the observed glycine conservation is not required for this essential processing step.

A clearer picture of the ER translocon complex.

The endoplasmic reticulum (ER) translocon complex is the main gate into the secretory pathway, facilitating the translocation of nascent peptides into the ER lumen or their integration into the lipid membrane. Protein biogenesis in the ER involves additional processes, many of them occurring co-translationally while the nascent protein resides at the translocon complex, including recruitment of ER-targeted ribosome-nascent-chain complexes, glycosylation, signal peptide cleavage, membrane protein topogenesis and folding. To perform such varied functions on a broad range of substrates, the ER translocon complex has different accessory components that associate with it either stably or transiently. Here, we review recent structural and functional insights into this dynamically constituted central hub in the ER and its components. Recent cryo-electron microscopy (EM) studies have dissected the molecular organization of the co-translational ER translocon complex, comprising the Sec61 protein-conducting channel, the translocon-associated protein complex and the oligosaccharyl transferase complex. Complemented by structural characterization of the post-translational import machinery, key molecular principles emerge that distinguish co- and post-translational protein import and biogenesis. Further cryo-EM structures promise to expand our mechanistic understanding of the various biochemical functions involving protein biogenesis and quality control in the ER.

Evolution for improved secretion and fitness may be the selective pressures leading to the emergence of two NDM alleles

The New Delhi metallo-β-lactamase (NDM-1) mediates resistance to β-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-β -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new blaNDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some β-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness.

Escherichia coli Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.