The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is, therefore, intriguing that some reports document the presence of orphan transporters SLC35F1 and SLC35F6 within the endosomal and lysosomal system. Here, we compared the subcellular distribution of these proteins and found that they are concentrated in separate compartments; i.e., recycling endosomes for SLC35F1 and lysosomes for SLC35F6. Swapping the C-terminal tail of these proteins resulted in a switch of localization, with SLC35F1 being trafficked to lysosomes while SLC35F6 remained in endosomes. This suggested the presence of specific sorting signals in these C-terminal regions. Using site-directed mutagenesis, fluorescence microscopy, and cell surface biotinylation assays, we found that the EQERLL360 signal located in the cytoplasmic tail of human SLC35F6 is involved in its lysosomal sorting (as previously shown for this conserved sequence in mouse SLC35F6), and that SLC35F1 localization in the recycling pathway depends on two YXXΦ-type signals: a Y367KQF sequence facilitates its internalization from the plasma membrane, while a Y392TSL motif prevents its transport to lysosomes, likely by promoting SLC35F1 recycling to the cell surface. Taken together, these results support that some SLC35 members may function at different levels of the endosomal and lysosomal system.

SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.

N-degrons are short sequences located at protein N-terminus that mediate the interaction of E3 ligases (E3s) with substrates to promote their proteolysis. It is well established that N-degrons can be exposed following protease cleavage to allow recognition by E3s. However, our knowledge regarding how proteases and E3s cooperate in protein quality control mechanisms remains minimal. Using a systematic approach to monitor the protein stability of an N-terminome library, we found that proline residue at the third N-terminal position (hereafter \"P+3\") promotes instability. Genetic perturbations identified the dipeptidyl peptidases DPP8 and DPP9 and the primary E3s of N-degron pathways, UBR proteins, as regulators of P+3 bearing substrate turnover. Interestingly, P+3 UBR substrates are significantly enriched for secretory proteins. We found that secretory proteins relying on a signal peptide (SP) for their targeting contain a \"built-in\" N-degron within their SP. This degron becomes exposed by DPP8/9 upon translocation failure to the designated compartments, thus enabling clearance of mislocalized proteins by UBRs to maintain proteostasis.

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa.
RESULTS: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%.
CONCLUSIONS: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

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.

BACKGROUND: Signal peptide (SP) engineering has proven able to improve production of many proteins yet is a laborious process that still relies on trial and error. mRNA structure around the translational start site is important in translation initiation and has rarely been considered in this context, with recent improvements in in silico mRNA structure potentially rendering it a useful predictive tool for SP selection. Here we attempt to create a method to systematically screen candidate signal peptide sequences in silico based on both their nucleotide and amino acid sequences. Several recently released computational tools were used to predict signal peptide activity (SignalP), localization target (DeepLoc) and predicted mRNA structure (MXFold2). The method was tested with Bone Morphogenetic Protein 2 (BMP2), an osteogenic growth factor used clinically for bone regeneration. It was hoped more effective BMP2 SPs could improve BMP2-based gene therapies and reduce the cost of recombinant BMP2 production.
RESULTS: Amino acid sequence analysis indicated 2,611 SPs from the TGF-β superfamily were predicted to function when attached to BMP2. mRNA structure prediction indicated structures at the translational start site were likely highly variable. The five sequences with the most accessible translational start sites, a codon optimized BMP2 SP variant and the well-established hIL2 SP sequence were taken forward to in vitro testing. The top five candidates showed non-significant improvements in BMP2 secretion in HEK293T cells. All showed reductions in secretion versus the native sequence in C2C12 cells, with several showing large and significant decreases. None of the tested sequences were able to increase alkaline phosphatase activity above background in C2C12s. The codon optimized control sequence and hIL2 SP showed reasonable activity in HEK293T but very poor activity in C2C12.
CONCLUSIONS: These results support the use of peptide sequence based in silico tools for basic predictions around signal peptide activity in a synthetic biology context. However, mRNA structure prediction requires improvement before it can produce reliable predictions for this application. The poor activity of the codon optimized BMP2 SP variant in C2C12 emphasizes the importance of codon choice, mRNA structure, and cellular context for SP activity.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

Proteins containing domain of unknown function (DUF) are prevalent in eukaryotic genome. The DUF1216 proteins possess a conserved DUF1216 domain resembling to the mediator protein of Arabidopsis RNA polymerase II transcriptional subunit-like protein. The DUF1216 family are specifically existed in Brassicaceae, however, no comprehensive evolutionary analysis of DUF1216 genes have been performed. We performed a first comprehensive genome-wide analysis of DUF1216 proteins in Brassicaceae. Totally 284 DUF1216 genes were identified in 27 Brassicaceae species and classified into four subfamilies on the basis of phylogenetic analysis. The analysis of gene structure and conserved motifs revealed that DUF1216 genes within the same subfamily exhibited similar intron/exon patterns and motif composition. The majority members of DUF1216 genes contain a signal peptide in the N-terminal, and the ninth position of the signal peptide in most DUF1216 is cysteine. Synteny analysis revealed that segmental duplication is a major mechanism for expanding of DUF1216 genes in Brassica oleracea, Brassica juncea, Brassica napus, Lepidium meyneii, and Brassica carinata, while in Arabidopsis thaliana and Capsella rubella, tandem duplication plays a major role in the expansion of the DUF1216 gene family. The analysis of Ka/Ks (non-synonymous substitution rate/synonymous substitution rate) ratios for DUF1216 paralogous indicated that most of gene pairs underwent purifying selection. DUF1216 genes displayed a specifically high expression in reproductive tissues in most Brassicaceae species, while its expression in Brassica juncea was specifically high in root. Our studies offered new insights into the phylogenetic relationships, gene structures and expressional patterns of DUF1216 members in Brassicaceae, which provides a foundation for future functional analysis.

DeepLoc 2.0 is a popular web server for the prediction of protein subcellular localization and sorting signals. Here, we introduce DeepLoc 2.1, which additionally classifies the input proteins into the membrane protein types Transmembrane, Peripheral, Lipid-anchored and Soluble. Leveraging pre-trained transformer-based protein language models, the server utilizes a three-stage architecture for sequence-based, multi-label predictions. Comparative evaluations with other established tools on a test set of 4933 eukaryotic protein sequences, constructed following stringent homology partitioning, demonstrate state-of-the-art performance. Notably, DeepLoc 2.1 outperforms existing models, with the larger ProtT5 model exhibiting a marginal advantage over the ESM-1B model. The web server is available at https://services.healthtech.dtu.dk/services/DeepLoc-2.1.

Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv\'s mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.