Protein Secretion Pathway Research Articles

Engineering Saccharomyces cerevisiae for continuous secretory production of hEGF in biofilm.

Human epidermal growth factor (hEGF) plays a crucial role in promoting cell growth and has various clinical applications. Due to limited natural sources and the high cost of chemical synthesis, researchers are now exploring genetic engineering as a potential method for hEGF production. In this particular study, a novel hEGF expression system was developed using Saccharomyces cerevisiae. This system involved optimizing the promoter and signal peptide and deleting protease-coding genes PEP4, PRB1, and YAP3, overexpressing chaperones KAR2 and PDI1 in the protein secretion pathway, which led to a 2.01-fold increase in hEGF production compared to the wild type strain. Furthermore, biofilm-forming genes FLO11 and ALS3 were integrated to create a biofilm strain with adhesive properties. A biofilm-based immobilized continuous fermentation model was established to leverage the characteristics of this biofilm strain. Each batch of this model yielded 130mg/L of hEGF, with a production efficiency of 2.71mg/L/h - surpassing the production efficiency of traditional free fermentation (1.62mg/L/h). This study presents a promising fermentation model for efficient hEGF production based on biofilm characteristics, offering valuable insights for the application of biofilm fermentation in the production of small molecule peptides.

The signal peptide of BmNPV GP64 activates the ERAD pathway to regulate heterogeneous secretory protein expression

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems.

Paths of Least Resistance: Unconventional Effector Secretion by Fungal and Oomycete Plant Pathogens.

Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional endoplasmic reticulum (ER)-Golgi pathway, while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In Magnaporthe oryzae, the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in M. oryzae requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, which is a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in M. oryzae has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide Phytophthora infestans cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion.

Eukaryotic cells have developed intricate mechanisms for biomolecule transport, particularly in stressful conditions. This interdisciplinary study delves into unconventional protein secretion (UPS) pathways activated during starvation, facilitating the export of proteins bypassing most of the components of the classical secretory machinery. Specifically, we focus on the underexplored mechanisms of the GRASP's role in UPS, particularly in biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results show that liquid-liquid phase separation (LLPS) plays a key role in the coacervation of Grh1, the GRASP yeast homologue, under starvation-like conditions. This association seems a precursor to the Compartment for Unconventional Protein Secretion (CUPS) biogenesis. Grh1's self-association is regulated by electrostatic, hydrophobic, and hydrogen-bonding interactions. Importantly, our study demonstrates that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations. Additionally, we explore how the coacervate liquid-to-solid transition could impact cells' ability to return to normal post-stress states. Our findings offer insights into intracellular protein dynamics and cell adaptive responses to stress.

Identification and characterization of the COPII vesicle-forming GTPase Sar1 in Chlamydomonas.

Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane-bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII-mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site-directed mutagenesis to generate a dominant-negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER-Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER-to-Golgi protein secretory pathway in green algae.

C9orf72 controls hepatic lipid metabolism by regulating SREBP1 transport.

Sterol regulatory element binding transcription factors (SREBPs) play a crucial role in lipid homeostasis. They are processed and transported to the nucleus via COPII, where they induce the expression of lipogenic genes. COPII maintains the homeostasis of organelles and plays an essential role in the protein secretion pathways in eukaryotes. The formation of COPII begins at endoplasmic reticulum exit sites (ERES), and is regulated by SEC16A, which provides a platform for the assembly of COPII. However, there have been few studies on the changes in SEC16A protein levels. The repetitive expansion of the hexanucleotide sequence GGGGCC within the chromosome 9 open reading frame 72 (C9orf72) gene is a prevalent factor in the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we found that the absence of C9orf72 leads to a decrease in SEC16A protein levels, resulting in reduced localization of the guanine nucleotide exchange factor SEC12 at the ERES. Consequently, the small GTP binding protein SAR1 is unable to bind the endoplasmic reticulum normally, impairing the assembly of COPII. Ultimately, the disruption of SREBPs transport decreases de novo lipogenesis. These results suggest that C9orf72 acts as a novel role in regulating lipid homeostasis and may serve as a potential therapeutic target for obesity.

Genetic Engineering of Filamentous Fungi: Prospects for Obtaining Fourth-Generation Biological Products

Filamentous fungi exhibit unparalleled potential as cell factories for protein production, owing to their adeptness in protein secretion and remarkable proficiency in post-translational modifications. This review delineates the role of filamentous fungi in bio-input technology across different generations and explores their capacity to generate secondary metabolites. Our investigation highlights filamentous fungi as frontrunners in the production of bioactive compounds, emphasizing the imperative nature of elucidating their metabolic repertoire. Furthermore, we delve into common strategies for genetic transformation in filamentous fungi, elucidating the underlying principles, advantages, and drawbacks of each technique. Taking a forward-looking approach, we explore the prospects of genome engineering, particularly the CRISPR-Cas9 technique, as a means to propel protein secretion in filamentous fungi. Detailed examination of the protein secretion pathways in these fungi provides insights into their industrial applications. Notably, extensive research within the scientific community has focused on Aspergillus and Trichoderma species for the industrial production of proteins and enzymes. This review also presents practical examples of genetic engineering strategies aimed at augmenting enzyme secretion in filamentous fungi for various industrial applications. These findings underscore the potential of filamentous fungi as versatile platforms for protein production and highlight avenues for future research and technological advancement in this field.

ATP2C1 knockdown induces abnormal expressions of cytoskeletal and tight junction proteins mimicking Hailey-Hailey disease.

Background Hailey-Hailey disease (HHD) is a rare, autosomal dominant, hereditary skin disorder characterised by epidermal acantholysis. The HHD-associated gene ATPase calcium-transporting type 2C member 1 (ATP2C1) encodes the protein secretory pathway Ca2+ ATPase1 (SPCA1), playing a critical role in HHD pathogenesis. Aims We aimed to investigate the effect of ATP2C1 knockdown on keratinocytes that mimicked acantholysis in HHD. Methods Immunohistochemistry (IHC) was employed to evaluate the levels of cytoskeletal and tight junction proteins such as SPCA1, P-cofilin, F-actin, claudins, occludin, and zonula occludens 1 in the skin biopsies of patients with HHD. Subsequently, the expression of these proteins in cultured ATP2C1 knockdown keratinocytes was analysed using Western blotting and immunofluorescence. Furthermore, we assessed the proliferation, apoptosis, and intracellular Ca2+ concentrations in the ATP2C1-knocked keratinocytes. Results The results showed decreased levels of these proteins (SPCA1, P-cofilin, F-actin, claudins, occluding, and zonula occludens 1) in HHD skin lesions. Moreover, their levels decreased in human keratinocytes transfected with ATP2C1 short hairpin RNA, accompanied by morphological acantholysis. Furthermore, the proliferation and apoptosis of the keratinocytes, as well as intracellular calcium concentrations in these cells, were not affected. Limitations The limitations of this study are the absence of animal experiments and the failure to explore the relationship between skeletal and tight junction proteins. Conclusion The present study indicated that ATP2C1 inhibition led to abnormal levels of the cytoskeletal and tight junction proteins in the keratinocytes. Therefore, keratinocytes can mimic HHD-like acantholysis and serve as an in vitro model, helping develop treatment strategies against HHD.

Active secretion of IL-33 from astrocytes is dependent on TMED10 and promotes central nervous system homeostasis

Interleukin-33 (IL-33), secreted by astrocytes, regulates the synapse development in the spinal cord and hippocampus and suppresses autoimmune disease in the central nervous system (CNS). However, the mechanism of unconventional protein secretion of this cytokine remains unclear. In this study, we found that IFN-γ promotes the active secretion of IL-33 from astrocytes, and the active secretion of IL-33 from cytoplasm to extracellular space was dependent on interaction with transmembrane emp24 domain 10 (TMED10) via the IL-1 like cytokine domain in astrocytes. Knockout of Il-33 or its receptor St2 induced hippocampal astrocyte activation and depressive-like disorder in naive mice, as well as increased spinal cord astrocyte activation and polarization to a neurotoxic reactive subtype and aggravated passive experimental autoimmune encephalomyelitis (EAE). Our results have identified that IL-33 is actively secreted by astrocytes through the unconventional protein secretion pathway facilitated by TMED10 channels. This process helps maintain CNS homeostasis by inhibiting astrocyte activation.

QseC regulates chemotaxis, biofilm formation, motility, and virulence in Aeromonas veronii TH0426

Aeromonas veronii is an important fish and human pathogen that causes significant economic losses in aquaculture. The two-component system QseBC has been shown to regulate virulence in various pathogens, but its functional role in A. veronii is unclear. In this study, we constructed in-frame deletion mutants of qseC in A. veronii TH0426 and complemented strains to determine the effects on pathogenesis-related phenotypes and global gene expression. Deletion of qseC did not significantly impact growth curves or colony morphology compared to wild-type A. veronii TH0426. However, the ΔqseC mutant displayed impaired swimming motility, with flagella staining and TEM images showing severe flagella detachment compared to intact flagella in wild-type cells. Crystal violet assays and confocal microscopy revealed that deletion of qseC significantly increased biofilm formation in A. veronii, with the ΔqseC mutant forming 2.07 times more biofilm than wild-type. Altered structural parameters indicative of increased heterogeneity were observed in the ΔqseC biofilm compared to wild-type using ISA analysis. Furthermore, the ΔqseC mutant showed enhanced adhesion to and invasion of epithelioma papulosum cyprini (EPC) cells in vitro. Using a zebrafish infection model, the ΔqseC mutant exhibited attenuated virulence with an 8.5-fold increase in LD50 value compared to wild-type A. veronii TH0426. Bacterial load enumeration in the blood, liver, spleen, and kidney of ΔqseC-infected carp over a 48-h period revealed significantly reduced bacterial counts compared to wild-type. In vivo competition assays indicated the ΔqseC mutant had reduced fitness in carp tissues compared to wild-type A. veronii. RNA sequencing revealed 2364 differentially expressed genes in the ΔqseC mutant compared to wild-type strain TH0426. KEGG pathway enrichment analysis indicated these genes were involved in chemotaxis, flagellar assembly, biofilm formation, protein secretion, and other pathways. Overall, this study demonstrates that QseC plays a vital regulatory role in controlling chemotaxis, flagellar motility, biofilm formation and virulence in the fish and human pathogen A. veronii.

Abstract 6459: Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data

Abstract Background: Up to 85% of lung cancer patients have non-small cell lung cancer (NSCLC). Standard of care for NSCLC includes immune checkpoint inhibitors (ICI) and/or platinum-based chemotherapy (CT). However, therapy success is limited with up to 70% of patients not responding to ICI. Mechanisms of resistance across treatments are not fully understood. In this study, we explore real-world data to characterize molecular and genomic changes in advanced NSCLC treated with ICI, ICI+CT, or CT. Methods: Stage III/IV NSCLC patients receiving first-line (1L) ICI, ICI+CT, or CT were identified from Tempus Labs, Inc.’s de-identified, multimodal real-world database. Patients’ solid tissue biopsies taken pre- or post-1L underwent next generation sequencing with the Tempus xT assays (DNAseq of 648 genes at 500x coverage and whole transcriptome RNAseq). Patients’ clinical and outcome data were abstracted from electronic health records. We analyzed changes in gene expression, pathway enrichment, estimated immune cell components, and somatic mutations in responders (complete/partial response) and non-responders (progression/stable disease) upon 1L treatment. Results: 1L non-responders (70%, 51%, and 47%) were identified across cohorts (143 ICI, 764 ICI+CT, and 228 CT, respectively). 58% of pre-1L biopsies were taken from distal metastatic tissues. Distinct pathways altered post-1L include the downregulated E2F target pathway in CT cohort, and upregulated TNFα/NFκB signaling and the hypoxia and protein secretion pathways in ICI cohort. Significant (FDR < 0.05) augmented expression was observed for 90, 23, and 65 genes in non-responders vs. responders post ICI, ICI+CT, and CT, respectively. RNAseq deconvolution reveals decreased plasma and CD4+ activated T cell fraction in metastatic biopsies, whereas increased CD4+ activated T cell and monocyte fraction was seen after treatment only in ICI cohort. Genes with oncogenic driver mutations reported in NSCLC were detected including TP53 (68%), KRAS (30%), STK11 (13%), EGFR (11%), CDKN2A (10%), KEAP1 (9%), and NF1 (7%). Of the 250 mutated genes, 72 (29%) were commonly not detected post-1L, whereas 38, 3, and 8 unique genes with loss of detectable mutations were identified post-1L of ICI, ICI+CT, and CT, respectively. Since loss of detectable mutations is a sign of loss of subpopulations of cancer cells hosting such mutations that succumb to therapy, these genetic mutations could be used to select patients who can benefit from such therapy. Conclusions: Distinct molecular characteristics including tumor immune components and genomic alterations are associated with 1L response in advanced NSCLC. Real-world data analyses provide opportunity to explore distinct mechanisms of therapy resistance and identify potential biomarkers of therapy response for further validation in clinical studies. Citation Format: Xiaoyong Fu, Jordan Kardos, Paola Correa, Min Wang, Li, Jackson Egen, Shahed Iqbal. Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6459.

Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

ER exit in physiology and disease.

The biosynthetic secretory pathway is comprised of multiple steps, modifications and interactions that form a highly precise pathway of protein trafficking and secretion, that is essential for eukaryotic life. The general outline of this pathway is understood, however the specific mechanisms are still unclear. In the last 15 years there have been vast advancements in technology that enable us to advance our understanding of this complex and subtle pathway. Therefore, based on the strong foundation of work performed over the last 40years, we can now build another level of understanding, using the new technologies available. The biosynthetic secretory pathway is a high precision process, that involves a number of tightly regulated steps: Protein folding and quality control, cargo selection for Endoplasmic Reticulum (ER) exit, Golgi trafficking, sorting and secretion. When deregulated it causes severe diseases that here we categorise into three main groups of aberrant secretion: decreased, excess and altered secretion. Each of these categories disrupts organ homeostasis differently, effecting extracellular matrix composition, changing signalling events, or damaging the secretory cells due to aberrant intracellular accumulation of secretory proteins. Diseases of aberrant secretion are very common, but despite this, there are few effective therapies. Here we describe ER exit sites (ERES) as key hubs for regulation of the secretory pathway, protein quality control and an integratory hub for signalling within the cell. This review also describes the challenges that will be faced in developing effective therapies, due to the specificity required of potential drug candidates and the crucial need to respect the fine equilibrium of the pathway. The development of novel tools is moving forward, and we can also use these tools to build our understanding of the acute regulation of ERES and protein trafficking. Here we review ERES regulation in context as a therapeutic strategy.

Molecular mechanisms of saliva secretion and hyposecretion.

The exocrine salivary gland secretes saliva, a fundamental body component to maintain oral homeostasis. Saliva is composed of water, ions, and proteins such as amylase, mucins, and immunoglobulins that play essential roles in the digestion of food, lubrication, and prevention of dental caries and periodontitis. An increasing number of people experience saliva hyposecretion due to aging, medications, Sjögren's syndrome, and radiation therapy for head and neck cancer. However, current treatments are mostly limited to temporary symptomatic relief. This review explores the molecular mechanisms underlying saliva secretion and hyposecretion to provide insight into putative therapeutic targets for treatment. Proteins implicated in saliva secretion pathways, including Ca2+ -signaling proteins, aquaporins, soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and tight junctions, are aberrantly expressed and localized in patients with saliva hyposecretion, such as Sjögren's syndrome. Analysis of studies on the mechanisms of saliva secretion and hyposecretion suggests that crosstalk between fluid and protein secretory pathways via Ca2+ /protein kinase C and cAMP/protein kinase A regulates saliva secretion. Impaired crosstalk between the two secretory pathways may contribute to saliva hyposecretion. Future research into the detailed regulatory mechanisms of saliva secretion and hyposecretion may provide information to define novel targets and generate therapeutic strategies for saliva hyposecretion.

An Overview of Protein Secretion in Plant Cells.

Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide are translocated into the endoplasmic reticulum and transported to the extracellular region via the endomembrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated findings and provide a comprehensive overview of protein secretion pathways in plants.

Identification and Validation of a Prognostic Signature Based on Fibroblast Immune-related Genes to Predict the Prognosis and Therapeutic Response of renal clear cell carcinoma by Integrated Analysis of Single-Cell and Bulk RNA Sequencing Data.

Background: The importance of fibroblasts in cancer progression is becoming more acknowledged, particularly the significance of their immune-related genes. However, the precise roles these genes play in fibroblasts throughout tumor development remains unclear. Exploring how these genes function in advancing kidney renal clear cell carcinoma (KIRC) could provide answers to these uncertainties. Material and method: The Cancer Genome Atlas (TCGA) database served as the source of data for KIRC patients. We distinguished fibroblast immune-related genes (FIGs), which are used to construct risk score. Further analysis conducted including enrichment analysis, assessment of tumor mutation burden (TMB), evaluation of tumor microenvironment (TME), analysis of immune cell infiltration, and drug sensitivity prediction. Result: The risk score using 6 FIGs effectively predicts the outcomes for KIRC patients. Nomogram which is based on the risk score and clinical data, demonstrated superior predictive performance compared to the version without the risk score. Enrichment analysis identified that coagulation pathway predominates in high-risk group, the protein secretion pathway is prevalent in low-risk patients' cohort. The adverse prognosis in high-risk patient cohort could be linked to an elevated TMB. TME analysis showed that high-risk group's tumor tissues contain more immune and stromal cells. Furthermore, the amount of regulatory T cells increases with the risk score. Low-risk group response better to immunotherapy. Finally, RT-qPCR confirmed the differential expression of FIGs in KIRC patients. Conclusion: This risk score and nomogram are valuable tools assessing KIRC patients' prognosis. Poorer prognosis in high-risk categories may have relationship with activation of coagulation pathway and a higher TMB.

Significance of RNA Processing Factors in Prognosis and Treatment of Hepatocellular Carcinoma: Implications for Precision Medicine

Hepatocellular carcinoma (HCC) is a malignant tumor with heterogenous nature and high mortality worldwide. RNA processing has been implicated in tumorigenesis, progression, and prognosis to develop early diagnosis and targeted therapy for cancer, including HCC. This study aimed to identify and validate a novel RNA processing gene signature for predicting HCC prognosis. Transcriptome and clinical data of HCC samples were obtained from Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), Gene Expression Omnibus databases, and RNA processing factors from the AmiGO database. The least absolute shrinkage and selection operator and multivariate Cox regression analysis were performed to examine prognostic gene signatures. Correlation analysis of prognostic models with clinical features and HallMark pathway-based Genomic Variation Analysis enrichment analysis were performed. We identified seven distinguished RNA processing factors to build the prognostic model for HCC patients. ROC curve analysis of the TCGA-HCC, ICGC-HCC, and GSE76427 cohorts determined the ability to predict prognosis through a sevengene-based model (AUC > 0.7). A nomogram to forecast the overall survival among HCC patients was established. In addition, 7 cell types (CD3+ cells, CD8+ T cells, macrophage/monocyte, monocyte, myeloid dendritic cells, neutrophils and cancer-associated fibroblast) significantly differed between the two risk groups. Furthermore, enrichment analysis showed significant enrichment in fatty acid metabolism, lipogenesis, pancreatic beta cells, and bile acid metabolism in the low-risk group, while in the high-risk group, DNA repair, protein secretion, and mitotic pathways were significantly enriched. Analyzing the Tumor Immune Dysfunction and Exclusion results demonstrated that immune checkpoint blockade therapy was poorly efficacious in the high-risk group. This study constructed and validated a novel prognostic signature related to RNA processing factors in HCC, improving therapeutic strategies for HCC. Building such prognostic signatures could pave the way for developing targeted therapy and precision medicine in HCC.

Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.

Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis.