Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage

ABSTRACT NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro. Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production. IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.

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Precise gene editing paves the way for derivation ofMannheimia haemolyticaleukotoxin-resistant cattle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613428113 ◽

2016 ◽

Vol 113 (46) ◽

pp. 13186-13190 ◽

Cited By ~ 13

Author(s):

Sudarvili Shanthalingam ◽

Ahmed Tibary ◽

Jonathan E. Beever ◽

Poothapillai Kasinathan ◽

Wendy C. Brown ◽

...

Keyword(s):

Signal Peptide ◽

Gene Editing ◽

Amino Acid Position ◽

Economic Loss ◽

Site Directed Mutagenesis ◽

Signal Peptidase ◽

Target Cells ◽

Cattle Industry ◽

Peptide Cleavage ◽

The Us

Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the β subunit of β2integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted byMannheimia(Pasteurella)haemolyticabinds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(‒5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(‒5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant toM. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.

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

10.1101/2020.11.11.378711 ◽

2020 ◽

Author(s):

A. Manuel Liaci ◽

Barbara Steigenberger ◽

Sem Tamara ◽

Paulo Cesar Telles de Souza ◽

Mariska Gröllers-Mulderij ◽

...

Keyword(s):

Active Site ◽

Catalytic Triad ◽

Signal Peptidase ◽

Structural Proteomics ◽

Signal Peptides ◽

Peptide Cleavage ◽

Atomic Structures ◽

Membrane Complex ◽

Human Signal ◽

Exquisite Specificity

AbstractThe 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.

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

Microbiology ◽

10.1099/mic.0.000937 ◽

2020 ◽

Vol 166 (8) ◽

pp. 759-776 ◽

Cited By ~ 4

Author(s):

Nathalie Dautin ◽

Manuela Argentini ◽

Niloofar Mohiman ◽

Cécile Labarre ◽

David Cornu ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Signal Peptide ◽

Type Species ◽

Signal Peptidase ◽

Covalent Attachment ◽

Post Translational Modification ◽

Content Type ◽

Peptide Cleavage ◽

Link Type ◽

Membrane Anchoring

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.

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