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Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to β-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased β-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of β-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate β-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating β-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment.

Aim. To study the effect of substance P on the blood serum glycoproteins in experimental animals with different stress-resistance profiles under technogenic rotating electric field. Methods. The level of sialic acids, mucoproteins, fucose, and -L-fucosidase was determined in the blood serum of 72 noninbred white male rats before (control) and on the 10th and 20th day of exposure to a technogenic rotating electric field (REF), as well as under the combination of technogenic rotating electric field and substance P injection at the same time. To determine the stress resistance, the animals were tested using the open field method. Animals were divided into groups based on the tests data obtained: stress-resistant, not stress-resistant and ambivalent. Results. On the 10th day of technogenic rotating electric field action, the level of sialic acids, fucose, and -L-fucosidase activity increased in all animals. The concentration of mucoproteins tended to decrease. On the 20th day, the sialic acids content remained elevated compared with the control in all groups. The content of mucoproteins decreased in stress-resistant, not stress-resistant and restored to the control level in ambivalent compared with those on the 10th day. On the 20th day, fucose concentration reached control values in stress-resistant and ambivalent animals and decreased in not stress-resistant. On the 10th day of the combined exposure, the concentration of sialic acids, mucoproteins, fucose, -L-fucosidase was reduced in all animals compared with the 10th day of technogenic rotating electric field action. On the 20th day of the combined exposure, the values of the studied parameters remained reduced in all groups of animals compared with those on the 20th day of isolated technogenic rotating electric field action. Conclusion. The substance P injection limits the effects of technogenic rotating electric field on the metabolism of carbohydrate-containing biopolymers in blood serum in all groups of animals, as can be seen by a decrease in the level of sialic acids, fucose, and low enzymatic activity of -L-fucosidase under combined exposure.

A alfa-1-glicoproteina ácida (AGP) e a mucoproteí­na são proteí­nas de fase inflamatória que aumentam suas concentrações plasmáticas quando apresentam um quadro de resposta ao estado inflamatório, representando um mecanismo de defesa do organismo. O objetivo do estudo foi avaliar o perfil dessas proteí­nas em pacientes com PCM crônica tratados com sulfametoxazol-trimetoprim (SMX-TMP) e associar os resultados encontrados com dados epidemiológicos, fatores de risco, sintomas, evolução da doença, e desfecho do tratamento. Nos métodos adotados foram analisados os prontuários de 244 pacientes com PCM crônica no perí­odo de 1998 a 2014. Destes, 134(54,92%) pacientes fizeram exames bioquí­micos das proteí­nas inflamatórias durante o curso da doença. Predominaram pacientes adultos de 30 a 50 anos 73(54,48%), tabagistas 123(91,79%), alcoólicos 60(44,78%). Como resultado obteve-se a diminuição das proteí­nas inflamatórias após o tratamento (p= 0,01803). Concluindo que a AGP e a mucoproteí­nas são úteis como marcadores do efeito de terapia e da involução inflamatória.

Acute pancreatitis (AP) is an inflammatory gastrointestinal disorder affecting the pancreas. Previous study reported that tetraspanin 1 (TSPAN1) expression was significantly upregulated in the pancreas of AP patients. However, the underlying molecular mechanism of TSPAN1 in the pathogenesis of AP remains unclear. Thus, the aim of the present study was to investigate the potential role of TSPAN1 in development of AP. RT-qPCR was carried out to quantify the relative mRNA levels of TSPAN1 and anterior gradient-2 (AGR2). The CCK-8 assay was used to detect the cell viability. The TUNEL assay was performed to visualize the apoptotic cells. Western blot was performed to determine the expressions of proteins related to endoplasmic reticulum (ER) stress and apoptosis. ELISA kits were adopted to detect the concentration of inflammatory cytokines including TNF-α and IL-6. Finally, immunoprecipitation (IP) was used to verify the interaction between TSPAN1 and AGR2. TSPAN1 was upregulated in serum of AP patients and AP cell models. TSPAN1 silencing promoted the cell proliferation and inhibited inflammatory response in cerulein-induced AR42J cells. Moreover, TSPAN1 induced endoplasmic reticulum stress by binding AGR2. Interestingly, the overexpression of AGR2 abolished the effects of TSPAN1 silencing on cell proliferation and inflammatory response in cerulein-induced AR42J cells. In summary, TSPAN1 silencing protects against cerulein-induced pancreatic acinar cell injury through inhibiting ER stress-mediated by AGR2. Hence, TSPAN1 may serve as a promising therapeutic target for AP treatment.

In this study, an ultrasonic-extracted polysaccharide (nCPTP-55) was obtained with the highest yield (61.08%, w/w) from tamarind pulp, which consisted chiefly of total sugar (85.98%, w/w) with few protein (2.10%, w/w). Monosaccharide analysis showed nCPTP-55 was mainly composed of arabinose (39.19 mol%) and glucose (50.48 mol%) with negligible GlcA (2.05 mol%), indicating the neutral nature of nCPTP-55, which was further elucidated structurally via GC–MS and NMR, i.e., an arabinoglucan composed of →3)-β-D-Glcp-(1→ backbone with only T-α-L-Araf-(1→ branched at O-4 (27.82%) and O-6 (39.99%), resulting in relatively high A/G ratio (0.68–0.70). Based on MM2 minimized energy, the 3D schematic structures of nCPTP-55 could be considered as structural basis for its conformational behavior, which was preliminarily estimated via HPSEC-MALLS as between compact sphere and loosely hyper-branched chain (ρ = 0.84). Therefore, the relationship between molecular structure and conformational behavior was basically established for nCPTP-55, which was in a bid to have a better knowledge of its structure-property and structure-bioactivity relationships potentially required for more applications in food, cosmetic and pharmaceutical fields.

It has been previously established that hypoxia leads to tumor development, treatment resistance, and a poor prognosis. Under oxygen deprivation, hypoxia-inducible factors (HIFs) are stimulated to activate the genes necessary for tumor development in a low-oxygen environment. These genes encode regulators of angiogenesis, epithelial-mesenchymal transition, and cellular metabolism. A disulfide isomerase, anterior gradient 2 (AGR2), has been shown to increase hypoxia-inducible factor 1, alpha subunit (HIF-1α) stability in breast cancer. Our goal was to determine if AGR2 affects the level of transcription factor hypoxia-inducible factor 2, alpha subunit (HIF-2α). As a model, we used the clear cell renal cell carcinoma (ccRCC) cell line Caki-1. The cells were transduced with lentiviral vector (Tet-On) encoding AGR2. After induction of AGR2 expression, cells were grown under either hypoxic (0.5% O2 ) or normoxic (21% O2 ) conditions. Our data showed that AGR2 upregulated both HIF-1α and HIF-2α expression in Caki-1 cells increasing the expression of HIF-activated genes (glucose transporter 1, phosphoglycerate kinase 1, vascular endothelial growth factor A, and transforming growth factor-alpha) under the hypoxic conditions. Under the normoxic conditions, AGR2 strongly activated CCAAT-enhancer binding protein beta (C/EBPβ). Upregulation of C/EBPβ correlated with increased expression and secretion of the interleukin-6 and interleukin-8, inducing angiogenesis and inflammation in Caki-1 cells. In summary, our studies revealed that AGR2 has essential functions in ccRCC progression through upregulation of C/EBPβ and HIF-2α expressions, which affects cell signaling and metabolism.

We developed a new system that causes specific degradation of type II arabinogalactans by dexamethasone-inducible expression of a fungal exo-β-1,3-galactanase in Arabidopsis, producing severe tissue disorganization in the hypocotyl and cotyledons., Arabinogalactan-proteins (AGPs) are a family of plant extracellular proteoglycans implicated in many physiological events. AGP is decorated with type II arabinogalactans (AGs) consisting of a β-1,3-galactan backbone and β-1,6-galactan side chains, to which other sugars are attached. Based on the fact that a type II AG-specific inhibitor, β-Yariv reagent, perturbs growth and development, it has been proposed that type II AGs participate in the regulation of cell shape and tissue organization. However, the mechanisms by which type II AGs participate have not yet been established. Here, we describe a novel system that causes specific degradation of type II AGs in Arabidopsis, by which a gene encoding a fungal exo-β-1,3-galactanase that specifically hydrolyzes β-1,3-galactan backbones of type II AGs is expressed under the control of a dexamethasone-inducible promoter. Dexamethasone treatment increased the galactanase activity, leading to a decrease in Yariv reagent-reactive AGPs in transgenic Arabidopsis. We detected the typical oligosaccharides released from type II AGs by Il3GAL in the soluble fraction, demonstrating that Il3GAL acted on type II AG in the transgenic plants. Additionally, this resulted in severe tissue disorganization in the hypocotyl and cotyledons, suggesting that the degradation of type II AGs affected the regulation of cell shape.

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

Root hairs (RHs) develop from specialized epidermal trichoblast cells, whereas epidermal cells that lack RHs are known as atrichoblasts. The mechanism controlling RH cell fate is only partially understood.RH cell fate is regulated by a transcription factor complex that promotes the expression of the homeodomain protein GLABRA 2 (GL2), which blocks RH development by inhibiting ROOT HAIR DEFECTIVE 6 (RHD6). Suppression of GL2 expression activates RHD6, a series of downstream TFs including ROOT HAIR DEFECTIVE 6 LIKE‐4 (RSL4) and their target genes, and causes epidermal cells to develop into RHs. Brassinosteroids (BRs) influence RH cell fate. In the absence of BRs, phosphorylated BIN2 (a Type‐II GSK3‐like kinase) inhibits a protein complex that regulates GL2 expression.Perturbation of the arabinogalactan peptide (AGP21) in Arabidopsis thaliana triggers aberrant RH development, similar to that observed in plants with defective BR signaling. We reveal that an O‐glycosylated AGP21 peptide, which is positively regulated by BZR1, a transcription factor activated by BR signaling, affects RH cell fate by altering GL2 expression in a BIN2‐dependent manner.Changes in cell surface AGP disrupts BR responses and inhibits the downstream effect of BIN2 on the RH repressor GL2 in root epidermis. Fil: Borassi, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Gloazzo Dorosz, Javier Anselmo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Ricardi, Martiniano María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Carignani Sardoy, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pol Fachin, Laercio. No especifíca; Fil: Marzol, Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Mangano, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rodríguez Garcia, Diana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Martinez Pacheco, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rondon Guerrero, Yossmayer del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Velásquez, Silvia Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Villavicencio, Bianca. Universidade Federal do Rio Grande do Sul ; Brasil Fil: Ciancia, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Seifert, Georg. University of Natural Resources and Life Science; Austria Fil: Verli, Hugo. Universidade Federal do Rio Grande do Sul ; Brasil Fil: Estevez, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Ontamalimab (SHP647) is a fully human, immunoglobulin G2, antihuman mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) monoclonal antibody being developed for the treatment of ulcerative colitis (UC) and Crohn's disease (CD). A population pharmacokinetic/pharmacodynamic (PK/PD) analysis was conducted using clinical phase 2 study data to evaluate the PK and PD of ontamalimab following subcutaneous administrations of 7.5, 22.5, 75, and 225 mg every 4 weeks in patients with moderate to severe UC or CD. A total of 440 patients with UC (n = 249; 56.6%) or CD (n = 191; 43.4%) were included in the analysis. A 2‐compartment model with parallel linear and nonlinear elimination adequately characterized concentration‐time profiles of ontamalimab. The apparent clearance and volume of distribution were 0.0127 L/h (0.305 L/day) and 6.53 L, respectively. Apparent clearance and volume of distribution were mainly dependent on baseline albumin and body weight, respectively. No differences in the PK properties of ontamalimab were observed between patients with UC or CD. The presence of antidrug antibodies did not impact the PK of ontamalimab. Nonlinear elimination occurred at very low concentrations and was unlikely to contribute to the elimination half‐life under steady‐state conditions. A linear PK/PD model described the relationship between ontamalimab and free MAdCAM‐1. Minimum concentrations of ontamalimab at steady state following 75 mg every 4 weeks were associated with >95% suppression of circulating free MAdCAM‐1. The PK/PD properties characterized support phase 3 testing in UC and CD.

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as ‘Picual’ undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in ‘Picual’ olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 β-galactoside α2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma (MM). Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a per-acetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of MM. However, 3Fax-Neu5Ac treatment of MM cells in vitro did not reverse bortezomib resistance conferred by bone marrow (BM) stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the BM. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective BM microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to BM-resident MM cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.

The anterior gradient-2 (AGR2) is an endoplasmic reticulum (ER)-resident protein belonging to the protein disulfide isomerase family that mediates the formation of disulfide bonds and assists the protein quality control in the ER. In addition to its role in proteostasis, extracellular AGR2 is responsible for various cellular effects in many types of cancer, including cell proliferation, survival, and metastasis. Various OMICs approaches have been used to identify AGR2 binding partners and to investigate the functions of AGR2 in the ER and outside the cell. Emerging data showed that AGR2 exists not only as monomer, but it can also form homodimeric structure and thus interact with different partners, yielding different biological outcomes. In this review, we summarize the AGR2 “interactome” and discuss the pathological and physiological role of such AGR2 interactions.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Anterior gradient protein 2 homolog (AGR2), an endoplasmic reticulum protein, is secreted in the tumor microenvironment. AGR2 is a member of the disulfide isomerase family, is highly expressed in multiple cancers, and promotes cancer metastasis. In this study, we found that etravirine, which is a non-nucleoside reverse transcriptase inhibitor, could induce AGR2 degradation via autophagy. Moreover, etravirine diminished proliferation, migration, and invasion in vitro. Moreover, in an orthotopic xenograft mouse model, the combination of etravirine and paclitaxel significantly suppressed cancer progression and metastasis. This drug may be a promising therapeutic agent for the treatment of ovarian cancer.

Arabinogalactan proteins (AGPs) are a superfamily of hydroxyproline-rich glycoproteins that are massively glycosylated, widely implicated in plant growth and development. No comprehensive analysis of the AGP gene family has been performed in Chinese cabbage (Brassica rapa ssp. chinensis). Here, we identified a total of 293 putative AGP-encoding genes in B. rapa, including 25 classical AGPs, three lysine-rich AGPs, 30 AG-peptides, 36 fasciclin-like AGPs (FLAs), 59 phytocyanin-like AGPs, 33 xylogen-like AGPs, 102 other chimeric AGPs, two non-classical AGPs and three AGP/extensin hybrids. Their protein structures, phylogenetic relationships, chromosomal location and gene duplication status were comprehensively analyzed. Based on RNA sequencing data, we found that 73 AGP genes were differentially expressed in the floral buds of the sterile and fertile plants at least at one developmental stage in B. rapa, suggesting a potential role of AGPs in male reproductive development. We further characterized BrFLA2, BrFLA28 and BrFLA32, three FLA members especially expressed in anthers, pollen grains and pollen tubes. BrFLA2, BrFLA28 and BrFLA32 are indispensable for the proper timing of pollen germination under high relative humidity. Our study greatly extends the repertoire of AGPs in B. rapa and reveals a role for three members of the FLA subfamily in pollen germination.

Charophyte green algae (CGA) are assigned to be the closest relatives of land plants and therefore enlighten processes in the colonization of terrestrial habitats. For the transition from water to land, plants needed significant physiological and structural changes, as well as with regard to cell wall composition. Sequential extraction of cell walls of Nitellopsis obtusa (Charophyceae) and Spirogyra pratensis (Zygnematophyceae) offered a comparative overview on cell wall composition of late branching CGA. Because arabinogalactan-proteins (AGPs) are considered common for all land plant cell walls, we were interested in whether these special glycoproteins are present in CGA. Therefore, we investigated both species with regard to characteristic features of AGPs. In the cell wall of Nitellopsis, no hydroxyproline was present and no AGP was precipitable with the β-glucosyl Yariv's reagent (βGlcY). By contrast, βGlcY precipitation of the water-soluble cell wall fraction of Spirogyra yielded a glycoprotein fraction rich in hydroxyproline, indicating the presence of AGPs. Putative AGPs in the cell walls of non-conjugating Spirogyra filaments, especially in the area of transverse walls, were detected by staining with βGlcY. Labelling increased strongly in generative growth stages, especially during zygospore development. Investigations of the fine structure of the glycan part of βGlcY-precipitated molecules revealed that the galactan backbone resembled that of AGPs with 1,3- 1,6- and 1,3,6-linked Galp moieties. Araf was present only in small amounts and the terminating sugars consisted predominantly of pyranosidic terminal and 1,3-linked rhamnose residues. We introduce the term 'rhamnogalactan-protein' for this special AGP-modification present in S. pratensis.

Previous studies have explored the association between protein-coding genes and microRNAs (miRNAs) in lung adenocarcinoma (LUAD). However, the influence of the miR-199a-3p/anterior gradient 2 (AGR2) axis in LUAD has not yet been fully explored. Therefore, this study aimed to examine the underlying roles of AGR2 and miR-199a-3p in the development of LUAD. The expression levels of miR-199a-3p and AGR2 in LUAD tissues and cells were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A luciferase assay was also performed to identify the interaction between AGR2 and miR-199a-3p. Moreover, the cell counting kit 8 (CCK-8), 5ʹ-bromo-2ʹ-deoxyuridine (BrdU), and adhesion assays were used along with flow cytometry to verify the malignancy of LUAD in vitro, while a xenograft tumor assay was performed to confirm the tumor growth in vitro. The findings showed a decrease in the expression of miR-199a-3p in LUAD. Additionally, miR-199a-3p overexpression inhibited the growth of LUAD cells in vitro and in vivo, while elevating the apoptosis rate of the cells. AGR2 knockdown had the same effect in the cells as that of miR-199a-3p overexpression. It was also found that miR-199a-3p directly targeted AGR2 in LUAD cells to suppress tumorigenesis. In conclusion, this study suggests that miR-199a-3p plays an anti-tumorigenic role in LUAD by targeting AGR2. Moreover, our study provides insights into the development of novel therapeutic targets for the treatment of LUAD.

The human gut microbiota (HGM) contributes to the physiology and health of its host. The health benefits provided by dietary manipulation of the HGM require knowledge of how glycans, the major nutrients available to this ecosystem, are metabolized. Arabinogalactan proteins (AGPs) are a ubiquitous feature of plant polysaccharides available to the HGM. Although the galactan backbone and galactooligosaccharide side chains of AGPs are conserved, the decorations of these structures are highly variable. Here, we tested the hypothesis that these variations in arabinogalactan decoration provide a selection mechanism for specific Bacteroides species within the HGM. The data showed that only a single bacterium, B. plebeius, grew on red wine AGP (Wi-AGP) and seaweed AGP (SW-AGP) in mono- or mixed culture. Wi-AGP thus acts as a privileged nutrient for a Bacteroides species within the HGM that utilizes marine and terrestrial plant glycans. The B. plebeius polysaccharide utilization loci (PULs) upregulated by AGPs encoded a polysaccharide lyase, located in the enzyme family GH145, which hydrolyzed Rha-Glc linkages in Wi-AGP. Further analysis of GH145 identified an enzyme with two active sites that displayed glycoside hydrolase and lyase activities, respectively, which conferred substrate flexibility for different AGPs. The AGP-degrading apparatus of B. plebeius also contained a sulfatase, BpS1_8, active on SW-AGP and Wi-AGP, which played a pivotal role in the utilization of these glycans by the bacterium. BpS1_8 enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health. IMPORTANCE Dietary manipulation of the HGM requires knowledge of how glycans available to this ecosystem are metabolized. The variable structures that decorate the core component of plant AGPs may influence their utilization by specific organisms within the HGM. Here, we evaluated the ability of Bacteroides species to utilize a marine and terrestrial AGP. The data showed that a single bacterium, B. plebeius, grew on Wi-AGP and SW-AGP in mono- or mixed culture. Wi-AGP is thus a privileged nutrient for a Bacteroides species that utilizes marine and terrestrial plant glycans. A key component of the AGP-degrading apparatus of B. plebeius is a sulfatase that conferred the ability of the bacterium to utilize these glycans. The enzyme enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health.

The increased use of nanoparticles (NP) in different industries inevitably results in their release into the environment. In such conditions, plants come into direct contact with NP. Knowledge about the uptake of NP by plants and their effect on different developmental processes is still insufficient. Our studies concerned analyses of the changes in the chemical components of the cell walls of Hordeum vulgare L. roots that were grown in the presence of gold nanoparticles (AuNP). The analyses were performed using the immunohistological method and fluorescence microscopy. The obtained results indicate that AuNP with different surface charges affects the presence and distribution of selected pectic and arabinogalactan protein (AGP) epitopes in the walls of root cells.

Novel molecular pinball machines of the plasma membrane control cytosolic Ca2+ levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca2+ channels; 4. auxin-efflux “PIN” proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants, “proton pinballs” eject Ca2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca2+. Freed Ca2+ ions flow down the electrostatic gradient through open Ca2+ channels into the cytosol, thus activating numerous Ca2+-dependent activities. Clearly, cytosolic Ca2+ levels depend on the activity of the proton pump, the state of Ca2+ channels and the size of the periplasmic AGP-Ca2+ capacitor; proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin. Auxin efflux carriers conveniently known as “PIN” proteins (null mutants are pin-shaped) pump auxin from cell to cell. Mechanosensitive Ca2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca2+. Cell expansion also triggers proton pump/pinball activity by the mechanotransduction of wall stress via Hechtian adhesion, thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth. Finally, the Ca2+ homeostasis of plants depends on cell surface external storage as a source of dynamic Ca2+, unlike the internal ER storage source of animals, where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarizes a sixty-year Odyssey.

The antigenic heterogeneity of the reticular framework of the white pulp and marginal zone is well documented in the human adult spleen. Immunostaining of α-smooth muscle actin characterizes the heterogeneity of the reticular framework of the white pulp and marginal zone. In the human spleen, the blood cells flow in an open circulation. T and B lymphocytes flow out from the arterial terminal, and migrate in the reticular framework. Homing of lymphocytes to lymphoid tissues is regulated by selective interactions between cell surface homing receptors and tissue vascular addressins at sites of lymphocyte recruitment from the blood. In the present study, mucosal addressin cell adhesion molecule-1 was selectively expressed on α-smooth muscle actin-positive reticular framework. The reticular framework may function in lymphocyte homing and segregation into the periarteriolar lymphoid sheath, lymph follicle and marginal zone.

Aims: AGR2 is a tissue-restricted member of the protein disulfide isomerase family that has attracted interest because it is highly expressed in a number of cancers, including gastroesophageal adenocarcinoma. The behavior of AGR2 was analyzed under oxidizing conditions, and an alkylation trapping and immunoprecipitation approach were developed to identify novel AGR2 interacting proteins. Results: The data show that AGR2 is induced in esophageal adenocarcinoma, where it participates in redox-responsive, disulfide-dependent complexes. AGR2 preferentially engages with MUC-5 as a primary client and is coexpressed with the acidic mucin in Barrett's esophagus and esophageal adenocarcinoma tissue. Innovation: New partner chaperones for AGR2 have been identified, including peroxiredoxin IV, ERp44, P5, ERp29, and Ero1α. AGR2 interacts with unexpected metabolic enzymes, including aldehyde dehydrogenase (ALDH)3A1, and engages in an alkylation-sensitive association with the autophagy receptor SQSTM1, suggesting a potential mechanism for the postendoplasmic reticulum targeting of AGR2 to mucin granules. Disulfide-driven AGR2 complex formation provides a framework for a limited number of client proteins to interact, rather than for the recruitment of multiple novel clients. Conclusion: The extended AGR2 interactome will facilitate the development of therapeutics to target AGR2/mucin pathways in esophageal cancer and other conditions, including chronic obstructive pulmonary disease.

Lymphocyte "homing", the physiologic trafficking of lymphocytes from the circulation to secondary lymphoid organs, is regulated by sequential adhesive interactions between lymphocytes and endothelial cells that constitute high endothelial venules (HEVs). Initial lymphocyte "rolling" is mediated by relatively weak, transient adhesive interactions between L-selectin expressed on lymphocytes and sulfated mucin-type O-glycans expressed on HEVs. Keratan sulfate galactose (Gal)-6-O-sulfotransferase (KSGal6ST) catalyzes 6-O-sulfation of Gal in keratan sulfate glycosaminoglycan chains but also transfers sulfate to Gal in much shorter glycan chains, such as sialylated N-acetyllactosamine (LacNAc)-capped O-glycans. In mice, KSGal6ST is reportedly expressed in HEVs and functions in synthesizing 6-sulfo Gal-containing O-glycans on HEVs. However, in humans, the presence of 6-sulfo Gal-containing O-glycans on HEVs is not reported. Employing the newly developed monoclonal antibody 297-11A, which recognizes non-sialylated terminal 6'-sulfo LacNAc, we demonstrate that sialyl 6'-sulfo (and/or 6,6'-disulfo) LacNAc-capped O-glycans are preferentially displayed on HEVs in human peripheral lymph nodes (PLNs) and to a lesser extent in mesenteric LNs (MLNs) but not in Peyer's patches (PPs). We also found that the scaffold protein mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed on HEVs in PPs and MLNs but not PLNs, was modified by 297-11A-positive sulfated glycans less efficiently than was CD34. Moreover, 297-11A-positive sulfated glycans were also displayed on HEV-like vessels induced in tumor-infiltrating lymphocyte (TIL) aggregates formed in various cancers. These findings collectively indicate that 297-11A-positive sulfated glycans potentially play a role in physiologic lymphocyte homing as well as in lymphocyte recruitment under pathologic conditions.