Binding Properties of Odorant-Binding Protein 4 of Tirathaba rufivena to Areca catechu Volatiles

Odorant-binding proteins (OBPs) play a key role in the olfactory system and are essential for mating and oviposition host selection. Tirathaba rufivena, a serious lepidopterous insect pest of the palm area in recent years, has threatened cultivations of Areca catechu in Hainan. Female-biased odorant-binding protein 4 of T. rufivena (TrufOBP4) expression was hypothesized to participate in the process of oviposition host recognition and localization. In this study, we cloned and analyzed the cDNA sequence of TrufOBP4. The predicted mature protein TrufOBP4 is a small, soluble, secretory protein and belongs to a classic OBP subfamily. Fluorescence binding assay results showed that TrufOBP4 had high binding abilities with the host plant volatiles, octyl methoxycinnamate, dibutyl phthalate, myristic acid and palmitic acid. These four components tend to dock in the same binding pocket based on the molecular docking result. The interactions and contributions of key amino acid residues were also characterized. This research provides evidence that TrufOBP4 might participate in the chemoreception of volatile compounds from inflorescences of A. catechu and can contribute to the integrated management of T. rufivena.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

Unconventional COPII-mediated secretory protein trafficking continues in the absence of the Sar1 GTPase

Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete membrane subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration of the coat subunit Sar1 and further stabilized by assembly of a multi-layer complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the unconventional carriers that form fail to efficiently deliver their contents to subsequent compartments in the secretory pathway. Instead, cargoes accumulate immediately adjacent to the perinuclear Golgi under these conditions, together with components of the COPII coat. Our findings highlight new mechanisms by which COPII promotes transport carrier biogenesis and strongly suggests that the Sar1 GTPase plays a critical role in transport carrier uncoating ahead of membrane fusion and secretory cargo delivery at acceptor compartments.

Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica—molecular background revealed by quantitative proteomics

In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg−1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo–keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided. Key points • Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica. • Enhanced abundance of HSPs and osmolytes is overweighted by limited translation. • Global proteome under Osm, Temp and Osm Temp treatments was studied.

Golgi Phosphoprotein 73: The Driver of Epithelial-Mesenchymal Transition in Cancer

Golgi phosphoprotein 73 (GP73, also termed as GOLM1 or GOLPH2) is a glycosylated protein residing on cis-Golgi cisternae and highly expressed in various types of cancer tissues. Since GP73 is a secretory protein and detectable in serum derived from cancer patients, it has been regarded as a novel serum biomarker for the diagnosis of different cancers, especially hepatocellular carcinoma (HCC). However, the functional roles of GP73 in cancer development are still poorly understood. In recent years, it has been discovered that GP73 acts as a multifunctional protein-facilitating cancer progression, and strikingly, it has been identified as a leading factor promoting epithelial-mesenchymal transition (EMT) of cancer cells and causing cancer metastasis. In this review, we have overviewed the latest findings of the functional roles of GP73 in elevating cancer progression, especially in facilitating EMT and cancer metastasis through modulating expression, transactivation, and trafficking of EMT-related proteins. In addition, unsolved research fields of GP73 have been lightened, which might be helpful to elucidate the regulatory mechanisms of GP73 on EMT and provide potential approaches in therapeutics against cancer metastasis.

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.

Cancer-associated adipocytes release FUCA2 to promote aggressiveness in TNBC

Cancer-associated adipocytes (CAAs) have been suggested to promote tumor progression. Yet, the role of CAAs in triple-negative breast cancer (TNBC) is poorly investigated. We compared the expression of secretory protein-encoding genes in CAAs and control adipocytes. The effect of key secretory protein(s) on TNBC cell behaviors was explored. CAAs expressed and secreted FUCA2 at greater levels than control adipocytes. When FUCA2 activity was blocked with a neutralizing antibody, TNBC cell proliferation and migration induced by CAA conditioned medium was impaired. In contrast, supplement of exogenous FUCA2 protein reinforced the proliferation, colony formation, and migration of TNBC cells. In vivo studies confirmed that FUCA2 exposure enhanced tumorigenesis and metastasis of TNBC cells. Mechanistic investigation revealed that FUCA2 induced TNBC aggressiveness through TM9SF3-dependent signaling. Depletion of TM9SF3 blocked CAA- and FUCA2-induced TNBC cell proliferation and migration. Compared to adjacent breast tissues, TNBC tissues had increased expression of TM9SF3. Moreover, high TM9SF3 expression was associated with advanced TNM stage, lymph node metastasis, and shorter overall survival of TNBC patients. Altogether, CAAs secrete FUCA2 to promote TNBC growth and metastasis through interaction with TM9SF3. Inhibition of TM9SF3 may represent a potential therapeutic strategy in the treatment of TNBC.

CC16 deficiency in the context of early life Mycoplasma pneumoniae infection results in augmented airway responses in adult mice.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16 -/- ) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults ( e arly l ife i nfection m odel; ELIM) and compared to adult mice infected for only three days ( a dult i nfection m odel; AIM). CC16 -/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16 -/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16 -/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16 -/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16 -/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

A Cytosolic Reductase Pathway is Required for Efficient N-Glycosylation of an STT3B-Dependent Acceptor Site

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A and STT3B-dependent glycosylation. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.