Xbp1-u and Xbp1-s from Nile tilapia (Oreochromis niloticus): Transcriptional profiling upon Streptococcus agalactiae infection and the potential role in B cell activation and differentiation.

X-box protein 1 (Xbp1), an essential transcription factor including an unstable form (Xbp1-u) and a stable form (Xbp1-s), plays an vital role in B cell activation and differentiation to plasma cells. In this study, we cloned and identified Xbp1-u gene from Nile tilapia (Oreochromis niloticus), containing 783 bp of nucleotide sequence encoding 260 amino acids. The deduced protein possesses a basic region leucine zipper domain (bZIP) and 26 ribonucleotides of OnXbp1-u transcript. Transcription analysis revealed OnXbp1-u and OnXbp1-s were widely distributed in all examined tissues, with a high expression in immune-related tissues. When stimulated with Streptococcus agalactiae in vivo, the expressions of OnXbp1-u and OnXbp1-s were significantly up-regulated in liver, spleen, head kidney, blood, skin and intestine. After in vitro challenge upon S.agalactiae, the similar up-regulations of OnXbp1-u and OnXbp1-s were also demonstrated in head kidney leukocytes. Moreover, the OnXbp1-u and OnXbp1-s could get involved in LPS-inducible B cell activation and (r)OnIL6-inducible B cell differentiation. Taken together, the results indicated that OnXbp1-u and OnXbp1-s might not only involved in the immune response against S. agalactiae challenge, but also in the B cell activation and differentiation in Nile tilapia.

New insights on human IRE1 tetramer structures based on molecular modeling.

Inositol-Requiring Enzyme 1α (IRE1α; hereafter IRE1) is a transmembrane kinase/ribonuclease protein related with the unfolded protein response (UPR) signaling. Experimental evidence suggests that IRE1 forms several three dimensional (3D) structural variants: dimers, tetramers and higher order oligomers, where each structural variant can contain different IRE1 conformers in different arrangements. For example, studies have shown that two sets of IRE1 dimers exist; a face-to-face dimer and a back-to-back dimer, with the latter considered the important unit for UPR signaling propagation. However, the structural configuration and mechanistic details of the biologically important IRE1 tetramers are limited. Here, we combine protein-protein docking with molecular dynamics simulations to derive human IRE1 tetramer models and identify a molecular mechanism of IRE1 activation. To validate the derived models of the human IRE1 tetramer, we compare the dynamic behavior of the models with the yeast IRE1 tetramer crystallographic structure. We show that IRE1 tetramer conformational changes could be linked to the initiation of the unconventional splicing of mRNA encoding X-box binding protein-1 (XBP1), which allows for the expression of the transcription factor XBP1s (XBP1 spliced). The derived IRE1 tetrameric models bring new mechanistic insights about the IRE1 molecular activation mechanism by describing the IRE1 tetramers as active protagonists accommodating the XBP1 substrate.

Systemic Delivery of Aptamer-Conjugated XBP1 siRNA Nanoparticles for Efficient Suppression of HER2+ Breast Cancer.

siRNA therapeutics as an emerging class of drug development is successfully coming to clinical utilization. The RNA-based therapy is widely utilized to explore the mechanism and cure a variety of gene-specific diseases. Tumor is an oncogene-driven disease; many genes are related to tumor progression and chemoresistance. Although human epidermal growth factor receptor 2 (HER2)-targeted monoclonal antibody therapy has dramatically improved the survival rate, chemotherapy remains essential to HER2-positive (HER2+) breast cancer patients. Recently, X-box binding protein 1 (XBP1) has been involved in triple-negative breast cancer (TNBC) chemoresistance and progression, but its function in HER2+ breast cancer is poorly explored. Here, we silenced XBP1 expression using RNase-resistant RNA nanoparticles (NPs). Intravenous injection of RNA NPs with HER2-specific aptamers resulted in strong binding to tumors but not to healthy tissues. XBP1 deletion by RNA NPs impaired angiogenesis and inhibited cell proliferation, significantly suppressed breast cancer growth, and promoted the sensitization of chemotherapy in an HER2+ breast cancer mouse model. Overall, these results reveal the function of XBP1 in HER2+ breast cancer development and chemoresistance and imply that targeting XBP1 by RNA NPs may offer an easy and promising strategy for a combination treatment of breast cancer in the future.

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method.

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

Structural and mutational analysis of the ribosome-arresting human XBP1u.

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.

The Metastable XBP1u Transmembrane Domain Defines Determinants for Intramembrane Proteolysis by Signal Peptide Peptidase.

Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that facilitate XBP1u turnover by an ER-associated degradation route that is dependent on signal peptide peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side-chain interactions near the scissile peptide bond with the enzyme's active site.

Merits and pitfalls of conventional and covalent docking in identifying new hydroxyl aryl aldehyde like compounds as human IRE1 inhibitors.

IRE1 is an endoplasmic reticulum (ER) bound transmembrane bifunctional kinase and endoribonuclease protein crucial for the unfolded protein response (UPR) signaling pathway. Upon ER stress, IRE1 homodimerizes, oligomerizes and autophosphorylates resulting in endoribonuclease activity responsible for excision of a 26 nucleotide intron from the X-box binding protein 1 (XBP1) mRNA. This unique splicing mechanism results in activation of the XBP1s transcription factor to specifically restore ER stress. Small molecules targeting the reactive lysine residue (Lys907) in IRE1α's RNase domain have been shown to inhibit the cleavage of XBP1 mRNA. Crystal structures of murine IRE1 in complex with covalently bound hydroxyl aryl aldehyde (HAA) inhibitors show that these molecules form hydrophobic interactions with His910 and Phe889, a hydrogen bond with Tyr892 and an indispensable Schiff-base with Lys907. The availability of such data prompted interest in exploring structure-based drug design as a strategy to develop new covalently binding ligands. We extensively evaluated conventional and covalent docking for drug discovery targeting the catalytic site of the RNase domain. The results indicate that neither computational approach is fully successful in the current case, and we highlight herein the potential and limitations of the methods for the design of novel IRE1 RNase binders.

Hac1p homologues from higher eukaryotes can improve the secretion of heterologous proteins in the yeast Pichia pastoris.

OBJECTIVES: To systematically explore the effects of overexpressing Hac1p homologues from different sources on protein secretion in Pichia pastoris system. RESULTS: Effects of Hac1p homologues encompassing P. pastoris (PpHac1p), S. cerevisiae (ScHac1p), Trichoderma reesei (TrHac1p) and Homo sapiens (HsXbp1), on secretion of three reporter proteins-ß-galactosidase, ß-mannanase and glucose oxidase were investigated. No individual Hac1p was optimal for all the enzymes. Rather, by testing a set of Hac1p, the secretory expression of each of the enzymes was improved. Notably, HsXbp1 overexpression improved ß-mannanase production from 73 to 108.5 U ß-mannanase mL-1 while PpHac1p had no impact in shake flask culture. Moreover, HsXbp1 led to 41 and 67% increases in ß-mannanase production in the single- and four-copy strain, respectively in 1-L laboratory fermenter. Transcription analysis of indicative chaperones suggested that HsXbp1 may cause a stronger and prolonged activation of the UPR target chaperone genes. CONCLUSION: Mammalian HsXbp1 worked better than yeast Hac1p in terms of improving ß-mannanase secretion in P. pastoris, and Hac1p screening may offer an effective strategy to engineer the secretion pathway of eukaryotic expression systems.

Histone deacetylase (HDAC) inhibitor ACY241 enhances anti-tumor activities of antigen-specific central memory cytotoxic T lymphocytes against multiple myeloma and solid tumors.

Histone deacetylases (HDAC) are therapeutic targets in multiple cancers. ACY241, an HDAC6 selective inhibitor, has shown anti-multiple myeloma (MM) activity in combination with immunomodulatory drugs and proteasome inhibitors. Here we show ACY241 significantly reduces the frequency of CD138+ MM cells, CD4+CD25+FoxP3+ regulatory T cells, and HLA-DRLow/-CD11b+CD33+ myeloid-derived suppressor cells; and decreases expression of PD1/PD-L1 on CD8+ T cells and of immune checkpoints in bone marrow cells from myeloma patients. ACY241 increased B7 (CD80, CD86) and MHC (Class I, Class II) expression on tumor and dendritic cells. We further evaluated the effect of ACY241 on antigen-specific cytotoxic T lymphocytes (CTL) generated with heteroclitic XBP1unspliced184-192 (YISPWILAV) and XBP1spliced367-375 (YLFPQLISV) peptides. ACY241 induces co-stimulatory (CD28, 41BB, CD40L, OX40) and activation (CD38) molecule expression in a dose- and time-dependent manner, and anti-tumor activities, evidenced by increased perforin/CD107a expression, IFN-γ/IL-2/TNF-α production, and antigen-specific central memory CTL. These effects of ACY241 on antigen-specific memory T cells were associated with activation of downstream AKT/mTOR/p65 pathways and upregulation of transcription regulators including Bcl-6, Eomes, HIF-1 and T-bet. These studies therefore demonstrate mechanisms whereby ACY241 augments immune response, providing the rationale for its use, alone and in combination, to restore host anti-tumor immunity and improve patient outcome.

Identification and characterization of HLA-A24-specific XBP1, CD138 (Syndecan-1) and CS1 (SLAMF7) peptides inducing antigens-specific memory cytotoxic T lymphocytes targeting multiple myeloma.

X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN)185-193 (I S P W I L A V L), XBP1 spliced (SP)223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F) and CS1240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24+ MM cells. The multipeptide-specific CTL included antigen-specific memory CD8+ T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.

CDK5-mediated phosphorylation of XBP1s contributes to its nuclear translocation and activation in MPP+-induced Parkinson's disease model.

Parkinson's disease (PD) is an irreversible and progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta. Growing evidence indicates that endoplasmic reticulum stress is a hallmark of PD; however, its exact contribution to the disease process remains poorly understood. Here, we used molecular biology methods and RNA-Seq analysis to explored an unexpected role of spliced X-Box binding protein 1 (XBP1s) in the nervous system. In this study, we determined that the IRE1α/XBP1 pathway is activated in MPP+-treated neurons. Furthermore, XBP1s was identified as a substrate of CDK5 and that the phosphorylation of XBP1s at the Ser61 residue enhances its nuclear migration, whereas mutation of the residue to alanine substantially reduces its nuclear translocation and activity. Importantly, phosphorylated XBP1s acts as a nuclear transcription factor for multiple target genes, including metabolic-related genes, FosB, and non-coding RNAs. Our findings confirm that the IRE1α/XBP1 pathway is activated in PD, and reveal a novel role of XBP1s in the pathogenesis of PD. This pathway may be a new therapeutic strategy for PD.

Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway.

Unconventional mRNA splicing on the endoplasmic reticulum (ER) membrane is the sole conserved mechanism in eukaryotes to transmit information regarding misfolded protein accumulation to the nucleus to activate the stress response. In metazoans, the unspliced form of X-box-binding protein 1 (XBP1u) mRNA is recruited to membranes as a ribosome nascent chain (RNC) complex for efficient splicing. We previously reported that both hydrophobic (HR2) and translational pausing regions of XBP1u are important for the recruitment of its own mRNA to membranes. However, its precise location and the molecular mechanism of translocation are unclear. We show that XBP1u-RNC is specifically recruited to the ER membrane in an HR2- and translational pausing-dependent manner by immunostaining, fluorescent recovery after photobleaching, and biochemical analyses. Notably, translational pausing during XBP1u synthesis is indispensable for the recognition of HR2 by the signal recognition particle (SRP), resulting in efficient ER-specific targeting of the complex, similar to secretory protein targeting to the ER. On the ER, the XBP1u nascent chain is transferred from the SRP to the translocon; however, it cannot pass through the translocon or insert into the membrane. Therefore, our results support a noncanonical mechanism by which mRNA substrates are recruited to the ER for unconventional splicing.

IRS1 deficiency protects ß-cells against ER stress-induced apoptosis by modulating sXBP-1 stability and protein translation.

Endoplasmic reticulum (ER) stress is among several pathological features that underlie ß-cell failure in the development of type 1 and type 2 diabetes. Adaptor proteins in the insulin/insulin-like-growth factor-1 signaling pathways, such as insulin receptor substrate-1 (IRS1) and IRS2, differentially impact ß-cell survival but the underlying mechanisms remain unclear. Here we report that ß-cells deficient in IRS1 (IRS1KO) are resistant, while IRS2 deficiency (IRS2KO) makes them susceptible to ER stress-mediated apoptosis. IRS1KOs exhibited low nuclear accumulation of spliced XBP-1 due to its poor stability, in contrast to elevated accumulation in IRS2KO. The reduced nuclear accumulation in IRS1KO was due to protein instability of Xbp1 secondary to proteasomal degradation. IRS1KO also demonstrated an attenuation in their general translation status in response to ER stress revealed by polyribosomal profiling. Phosphorylation of eEF2 was dramatically increased in IRS1KO enabling the ß-cells to adapt to ER stress by blocking translation. Furthermore, significantly high ER calcium (Ca(2+)) was detected in IRS1KO ß-cells even upon induction of ER stress. These observations suggest that IRS1 could be a therapeutic target for ß-cell protection against ER stress-mediated cell death by modulating XBP-1 stability, protein synthesis, and Ca(2+) storage in the ER.