Signal Peptide Research Articles

Fc engineered anti-virus therapeutic human IgG1 expressed in plants with altered binding to the neonatal Fc receptor.

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

The Practicality of Resistivity Method for Recognizing Vertically Distributed Anomalies Using Simulated Models

In brownfield sites, two proposed shallow horizontal targets were numerically modelled to mimic variable underground conditions; these targets were sequenced vertically in a different manner. Synthetic 2D electrical resistivity datasets were generated using the RES2DMOD and then inverted by using RES2DINVx64. These targets have different lengths, one with a higher and the second with a lower resistivity value relative to the background resistivity. The effectiveness of dipole-dipole, Wenner, and Wenner-Schlumberger configurations was tested by conducting a qualitative comparison. The inverted resistivity images suggest that the target sequence has a significant effect on the configuration detectability. The high resistivity target was conveniently recognized when it overlies a low resistivity target. Dipole-dipole configuration showed optimum results in recognizing complicated underground conditions.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Molecular characterization, antibacterial and immunoregulatory activities of liver-expressed antimicrobial peptide 2 in black rockfish, Sebastes schlegelii

LEAP2 (liver expression antimicrobial peptide 2), is an antimicrobial peptide widely found in vertebrates and mainly expressed in liver. LEAP2 plays a vital role in host innate immunity. In teleosts, a number of LEAP2 homologs have been reported, but their in vivo effects on host defense are still limited. In this study, a LEAP2 homolog (SsLEAP2) was identified from black rockfish, Sebastes schlegelii, and its structure, expression as well as biological functions were analyzed. The results showed that the open reading frame of SsLEAP2 is 300 bp, with a 5′- untranslated region (UTR) of 375 bp and a 3’ - UTR of 238 bp. The deduced amino acid sequence of SsLEAP2 shares the highest overall identity (96.97%) with LEAP2 of Sebastes umbrosus. SsLEAP2 possesses conserved LEAP2 features, including a signal peptide sequence, a prodomain and a mature peptide, in which four well-conserved cysteines formed two intrachain disulphide domain. The expression of SsLEAP2 was highest in liver and could be induced by experimental infection with Listonella anguillarum, Edwardsiealla piscicida and Rock bream iridovirus C1 (RBIV-C1). Recombinant SsLEAP2 (rSsLEAP2) purified from Escherichia coli was able to bind with various Gram-positive and Gram-negative bacteria. Further analysis showed that rSsLEAP2 could enhance the respiratory burst activity, and induce the expression of immune genes including interleukin 1-β (IL-1β) and serum amyloid A (SAA) in macrophages; additionally, rSsLEAP2 could also promote the proliferation and chemotactic of peripheral blood lymphocytes (PBLs). In vivo experiments indicated that overexpression of SsLEAP2 could inhibit bacterial infection, and increase the expression level of immune genes including IL-1β, tumor necrosis factor ligand superfamily member 13B (TNF13B) and haptoglobin (HP); conversely, knock down of SsLEAP2 promoted bacterial infection and decreased the expression level of above genes. Taken together, these results suggest that SsLEAP2 is a novel LEAP2 homolog that possesses apparent antibacterial activity and immunoregulatory property, thus plays a critical role in host defense against pathogens invasion.

A Bursaphelenchus xylophilus effector BxICD1 inducing plant cell death, concurrently contributes to nematode virulence and migration.

The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian.

Comparative study on the target sequences of thaumatin-like protein (TLP) and internal transcribed spacer (ITS) in the detection of corn component in edible oil by ladder-shape melting temperature isothermal amplification (LMTIA)

Single copy thaumatin-like protein (TLP) genes and multiple copy internal transcribed spacer (ITS) genes from the nucleus of corn were selected as targets, designed and screened primers. The impact of target sequence selection on ladder-shape melting temperature isothermal amplification (LMTIA) detection of corn components in edible oil from aspects such as specificity, sensitivity, and stability were studied. The results showed that the LMTIA system targeting TLP has an optimal reaction temperature of 58 °C and can detect a minimum of 100 pg/μL of corn DNA, and the LMTIA system established with ITS as the target has higher sensitivity, with an optimal temperature of 59 °C, and can detect corn DNA as low as 1 pg/μL. Both systems have good specificity, stable reactions, and no false positive results were observed. Conclusion: ITS is more suitable as a target sequence for LMTIA detection of corn components in edible oil.

High-Level Extracellular Expression of Hyaluronate Lyase HylP in Bacillus subtilis for Hyaluronan Degradation.

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40°C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40°C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.

Characterization of a novel γ-type carbonic anhydrase, Sjγ-CA2, in Saccharina japonica: Insights into carbon concentration mechanism in macroalgae

Carbonic anhydrase (CA) is a crucial component of CO2-concentrating mechanism (CCM) in macroalgae. In Saccharina japonica, an important brown seaweed, 11 CAs, including 5 α-, 3 β-, and 3 γ-CAs, have been documented. Among them, one α-CA and one β-CA were localized in the periplasmic space, one α-CA was found in the chloroplast, and one γ-CA was situated in mitochondria. Notably, the known γ-CAs have predominantly been identified in mitochondria. In this study, we identified a chloroplastic γ-type CA, Sjγ-CA2, in S. japonica. Based on the reported amino acid sequence of Sjγ-CA2, the epitope peptide for monoclonal antibody production was selected as 165 Pro-305. After purification and specificity identification, anti-SjγCA2 monoclonal antibody was employed in immunogold electron microscopy. The results illustrated that Sjγ-CA2 was localized in the chloroplasts of both gametophytes and sporophytes of S. japonica. Subsequently, immunoprecipitation coupled with LC-MS/MS analysis revealed that Sjγ-CA2 mainly interacted with photosynthesis-related proteins. Moreover, the first 65 amino acids at N-terminal of Sjγ-CA2 was identified as the chloroplast transit peptide by the transient expression of GFP-SjγCA2 fused protein in tabacco. Real-time PCR results demonstrated an up-regulation of the transcription of Sjγ-CA2 gene in response to high CO2 concentration. These findings implied that Sjγ-CA2 might contribute to minimizing the leakage of CO2 from chloroplasts and help maintaining a high concentration of CO2 around Rubisco.

Characterization of a membrane Fcγ receptor in largemouth bass (Micropterus saloumoides) and its response to bacterial challenge.

Fc receptors (FcRs), specific to the Fc portion of immunoglobulin (Ig), are required to regulate immune responses against pathogenic infections. However, FcγR is a member of FcRs family, whose structure and function remains to be elucidated in teleost fish. In this study, the FcγRII, from largemouth bass (Micropterus saloumoides), named membrane MsFcγRII (mMsFcγRII), was cloned and identified. The opening reading frame (ORF) of mMsFcγRII was 750 bp, encoding 249 amino acids with a predicted molecular mass of 27 kDa. The mMsFcγRII contained a signal peptide, two Ig domains, a transmembrane domain, and an intracellular region, which was highly homology with FcγR from other teleost fish. The mRNA expression analysis showed that mMsFcγRII was widely distributed in all tested tissues and with the highest expression level in spleen. After bacterial challenge, the expression of mMsFcγRII was significantly upregulated in vivo (spleen and head kidney), as well as in vitro (leukocytes from head kidney). The subcellular localization assay revealed that mMsFcγRII was mostly observed on the membrane of HEK293T cells which were transfected with mMsFcγRII overexpression plasmid. Flow cytometric analysis showed that natural mMsFcγRII protein was highly expressed in head kidney lymphocytes. Moreover, indirect immunofluorescence assay and pull-down assay indicated that mMsFcγRII could bind to IgM purified from largemouth bass serum. These results suggested that mMsFcγRII was likely to play an influential role in the immune response against pathogens and provided valuable insights for studying the function of FcRs in teleost.

Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

High-Level Expression and Biochemical Characterization of a Maltotetraose Amylase in Pichia pastoris X-33 for Maltotetraose Production.

Maltotetraose amylase, which catalyzes the hydrolysis of amylaceous polysaccharides into maltooligosaccharides with maltotetraose as the main product, is extensively used in the food industry. However, the lack of efficient expression system for maltotetraose amylase has hampered its production and application. In this study, high-level production of a maltotetraose amylase mutant (referred to as Pp-Mta∆CBM) from Pseudomonas saccharophila was achieved in Pichia pastoris X-33. First, the gene of maltotetraose amylase with the carbohydrate-binding module (CBM) removed was codon-optimized and cloned into the pPICZαA vector, followed by transformation into P. pastoris X-33 for expression. Using the promoter PAOX1 and signal peptide α-factor, high-level production of Pp-Mta∆CBM with minimal extracellular impurity proteins was achieved, resulting in an extracellular activity of 367.9 U/mL after 7 days of cultivation in shake flasks. Next, the expressed Pp-Mta∆CBM was purified and characterized. This recombinant enzyme was glycosylated and has maximum activity at 55 ℃ and pH 7.0. Its Km for soluble starch was 4.1g/L, and its kcat was 3237.6s-1. Finally, the Pp-Mta∆CBM was found to produce a maximum maltotetraose yield of 57.1% in the presence of 200g/L of substrate. The findings presented in this study demonstrate the efficient production of Pp-Mta∆CBM in P. pastoris, providing a new expression system for maltotetraose amylase and laying the foundation for its scale-up production and industrial application.

Identification and Expression Profile Analysis of WOX Family Genes in the Formation of Eucalyptus Adventitious Root

The WUSCHEL-related homeobox (WOX) gene family are key players in the rooting process. Eucalyptus is an important plant species of artificial forests in China. It is mainly grown through tissue culture of many excellent clonal materials, in which rooting is a key step. In the present study, by using the genome data of Eucalyptus grandis, Corymbia citriodora, E. pellita, and E. urophylla × E. grandis, the members of the eucalyptus WOX gene family were identified and analyzed by bioinformatics techniques. The eucalyptus WOX gene family members are unstable proteins, with 7 acidic proteins and 24 basic proteins, and no signal peptide region was detected. Subcellular localization prediction indicated that all these proteins are localized in the nucleus. Motif analysis showed that eucalyptus WOX genes share the same motifs. Phylogenetic tree and gene expression analyses revealed that the eucalyptus WOX genes are highly conserved during the evolution process. Moreover, the WOX protein sequences are also highly conserved within the species, with higher similarity between woody plants. The EupWOX gene showed tissue-specific expression, with EupWOX1 and EupWOX11 specifically expressed in the roots of E. urophylla × E. pellita clonal tissue culture during the late-stage rooting. This finding suggests that EupWOX1 may be a key regulatory gene induced by the root primordium and is critically related to the rooting rate. EupWOX1, EupWOX5, and EupWOX13 could be the key regulatory genes for adventitious root formation. EupWOX1, EupWOX5, and EupWOX13 could be the key regulatory genes for the elongation of adventitious roots and the growth of adventitious lateral roots. EupWOX5 and EupWOX13 could play a critical role, not only in the formation of adventitious roots and adventitious lateral roots of E. urophylla clonal tissue culture but also in the root elongation process. These results will help us understand the complexity of rooting in different lines and provide valuable information for future functional characterization of specific genes in eucalyptus clones.

Effect of signal peptide optimized by site-directed mutagenesis on the function of chimeric antigen receptor T cells

Signal peptides (SP) are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors (CAR), which is crucial for CAR-T cells. This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T. Firstly, CAR vectors targeting CD19 containing wild-type SP (SP-wtY) or two mutant SP (SP-muK or SP-muR) were constructed using gene synthesis and molecular cloning techniques. The successfully constructed vector was packaged with lentivirus, and T cells were infected. The transfection efficiency of T cells was detected by flow cytometry, while the killing effect on target cells was assessed using the calcein release method. The secretion levels of cytokines interferon-γ (IFN-γ) and interferon-α (TNF-α) were measured using enzyme linked immunosorbent assay (ELISA). The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation. After the transferring the lentivirus into T cells, the transfection efficiency of CD19-CAR carrying three signal peptides (SP-wtY, SP-muK, or SP-muR) were 33.9%, 35.5%, and 36.8%, respectively. Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells. When the ratio of effector to target was 10:1, the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups. In summary, this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19+ target cells. These findings provide a scientific basis the optimization and clinical application of CAR structure.

Unveiling the dynamic expression of PR-1 during Musa spp. infection by Fusarium oxysporum fsp. Cubense: a cloning and characterization study.

Pathogen-related proteins (PR) are pivotal in plant defense, combating diverse biotic and abiotic stresses. While multiple gene families contribute to banana resistance against Fusarium oxysporum f sp. cubense (Foc), Pseudocercospora eumusae, and Pratylenchus coffeae, the significance of PR-1 genes in defense is paramount. Three PR-1 genes, up-regulated under diverse biotic stresses, were cloned from both resistant and susceptible cultivars of Foc, P. eumusae, and P. coffeae. Molecular characterization, phylogenetic analysis, and docking studies with the Foc TR4 CP gene were conducted. Through transcriptomic and real-time studies, three PR-1 genes (Ma02_g15050, Ma02_g15060, and Ma04_g34800) from Musa spp. were identified. These genes exhibited significant up-regulation in resistant cultivars when exposed to Foc, P. eumusae, and P. coffeae. Cloning of these genes was successfully performed from both resistant and susceptible cultivars of Foc race 1 and TR4, P. eumusae, and P. coffeae. Distinct characteristics were observed among the PR-1 genes, with groups 1 and 2 being acidic with signal peptides, and group 3 being basic without signal peptides. All cloned PR-1 proteins belonged to the CAP superfamily (PF00188). Phylogenetic analysis revealed clustering patterns for acidic PR-1 proteins, and KEGG orthology showed associations with vital pathways, including MAPK signaling, plant hormone signal transduction, and plant-pathogen interaction. Secondary and tertiary structure analyses confirmed sequence conservation across studied species. Docking studies explored interactions between the cerato-platanin (CP) gene from Foc TR4 and Ma02_g15060 from banana, suggesting the potential hindrance of PR-1 antifungal activity through direct interaction. The findings underscore the crucial role of cloned PR-1 genes in banana plant defense mechanisms against a broad spectrum of biotic stresses. These genes, especially those in groups 1 and 2, hold promise as candidates for developing stress-tolerant banana cultivars. The study provides valuable insights into the molecular aspects of banana defense strategies, emphasizing the potential applications of PR-1 genes in enhancing banana resilience.

Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.

Real‐Time, Non‐Invasive Monitoring of Neuronal Differentiation Using Intein‐Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell‐Based Biosensors

AbstractReal‐time and non‐invasive monitoring of neuronal differentiation helps to increase understanding of neuronal development and develop stem cell therapies for neurodegenerative diseases. Conventional methods such as RT‐PCR, western blotting, and immunofluorescence (IF), lack single‐cell‐level resolution and require invasive procedures, fixation, and staining. These limitations hinder accurate monitoring progress of neural stem cell (NSC) differentiation and understanding its functions. Herein, a novel approach is reported to non‐invasively monitor neuronal differentiation in real‐time using cell‐based biosensors (CBBs) that detects hippocalcin, biomarker of neuronal differentiation. To construct the hippocalcin sensor proteins, two different hippocalcin bioreceptors are fused to each split‐intein, carrying split‐nuclear localization signal (NLS) peptides, respectively, and fluorescent protein is introduced as reporter. CBBs operated in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The NSC‐based biosensor shows fluorescence signal translocation only upon neuronal differentiation and not undifferentiated stem cells or glial cells. Furthermore, this approach allows monitoring of neural differentiation at earlier stages than detected using IF staining. It is believed that novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single‐cell‐level and providing a tool to evaluate how NSCs efficiently differentiate into neurons.

Genome-wide survey of peptides containing tyrosine sulfation (PSY) gene family and potential PSY specific miRNA revealed their role in plant development and diverse stress conditions in rice (Oryza sativa L.)

Signalling peptides play a vital role in regulating a wide range of aspects, including plant growth, development, and responses to various biotic and abiotic conditions. In recent times, small post-translationally altered peptides have garnered significant attention from plant biologists due to their crucial role as signalling molecules in various biological processes. Until now, the biological function of peptides containing Tyrosine Sulfation (PSY) gene family members has only been elucidated in Arabidopsis. However, their functions in plant developmental processes and their responses to different biotic and abiotic stress conditions remain elusive in other crops. Therefore, in this study, we identified 109 PSY genes in the rice genome for the first time. However, our detailed in silico analysis focused specifically on 7 OsPSY genes of the O. sativa Japonica Group (Nippobare). Phylogenetic analysis of PSY genes from various plant species has classified these genes into eight distinct groups, designated as Group I to VIII. Interestingly, the 7 OsPSY genes identified in rice were found to be distributed across all four groups including I, V, VI and VIII. In addition, closely linked groups of PSY genes showed more similarities in terms of gene structures, conserved motif distributions, and corresponding subcellular locations. Further, the Ka/Ks score analysis revealed that OsPSY genes have undergone significant purifying selection throughout their evolutionary history. Furthermore, multiple CAREs were discovered in the OsPSY promoters, suggesting their potential importance in regulating plant growth, responses to plant hormones, and multifactorial stress conditions. Additionally, the potential miRNAs that specifically targeted to OsPSY genes were identified and also examined their expression kinetics in different tissues and developmental stage. Out of the seven OsPSY genes, six OsPSY genes were found to be targeted by twelve miRNAs. Moreover the expression profile of OsPSY genes showed a differential expression pattern in various tissues and multifactorial stress conditions. Therefore, these findings serve as a valuable reference for further molecular studies aimed at enhancing the yield and stress tolerance of rice. The understanding the functions and regulatory mechanisms of the OsPSY genes can potentially lead to the development of targeted approaches to improve crop performance under various environmental conditions, ultimately contributing to agricultural productivity and global food security.

B. subtilis Sec and Srp Systems Show Dynamic Adaptations to Different Conditions of Protein Secretion.

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

Heterologous Expression and Characterization of a Novel Mesophilic Maltogenic α-Amylase AmyFlA from Flavobacterium sp. NAU1659.

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli. Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50°C in 50 mM phosphate buffer (pH 6.0). The Km and Vmax values of AmyFlA were respectively 3.15mg/ml and 566.36 µmol·ml-1·min-1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca2+, indicating that AmyFlA was a Ca2+-dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup.

Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences.

The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.