Type 1 Calreticulin Mutations Differentially Activate the IRE1α-XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. Here, we show that type 1 CALRdel52 but not type 2 CALRins5 mutations lead to activation of and dependency on the IRE1α-XBP1 pathway of the unfolded protein response (UPR). Mechanistically, we found that the loss of calcium binding residues in the type 1 mutant CALR protein directly impairs its calcium binding ability, which in turn leads to depleted ER calcium and subsequent activation of the IRE1α-XBP1 pathway. Using cell lines and primary MPN patient samples, we identified two novel transcriptional targets of XBP1 specific to type 1 CALRdel52-expressing cells - the anti-apoptotic protein BCL-2 and the calcium efflux channel IP3R. We show that BCL-2 acts downstream of XBP1 to promote survival in the face of depleted ER calcium, while IP3R is up-regulated downstream of XBP1 to promote continued ER calcium efflux in order to sustain IRE1α-XBP1 pathway activation and survival. We found that genetic or pharmacological inhibition of IRE1α-XBP1 signaling induced cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells. Moreover, we show that in vivo inhibition of IRE1α significantly abrogates type 1 mutant CALR-driven disease in a bone marrow transplantation model, but has no effect on type 2 mutant CALR-driven disease. This work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be exploited for therapeutic gain. Moreover, this study answers an enduring question regarding the functional consequence of the loss of calcium binding sites on the type 1 mutant CALR protein, and demonstrates how type 1 CALR mutant-expressing cells rewire the UPR, downstream calcium signaling, and apoptotic pathways to drive MPN. Figure 1 Figure 1. Disclosures Koschmieder: BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Karthos: Other: Travel support; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Abbvie: Other: Travel support; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); CTI: Membership on an entity's Board of Directors or advisory committees, Other; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Image Biosciences: Other: Travel support.

Huntingtin: A Protein with a Peculiar Solvent Accessible Surface

Taking advantage of the last cryogenic electron microscopy structure of human huntingtin, we explored with computational methods its physicochemical properties, focusing on the solvent accessible surface of the protein and highlighting a quite interesting mix of hydrophobic and hydrophilic patterns, with the prevalence of the latter ones. We then evaluated the probability of exposed residues to be in contact with other proteins, discovering that they tend to cluster in specific regions of the protein. We then found that the remaining portions of the protein surface can contain calcium-binding sites that we propose here as putative mediators for the protein to interact with membranes. Our findings are justified in relation to the present knowledge of huntingtin functional annotation.

Molecular characterization and expression analysis of annexin B3 and B38 as secretory proteins in Echinococcus granulosus

Background Cystic echinococcosis is a parasitic zoonotic disease, which poses a threat to public health and animal husbandry, and causes significant economic losses. Annexins are a family of phospholipid-binding proteins with calcium ion-binding activity, which have many functions. Methods Two annexin protein family genes [Echinococcus granulosus annexin B3 (EgAnxB3) and EgAnxB38] were cloned and molecularly characterized using bioinformatic analysis. The immunoreactivity of recombinant EgAnxB3 (rEgAnxB3) and rEgAnxB38 was investigated using western blotting. The distribution of EgAnxB3 and EgAnxB38 in protoscoleces (PSCs), the germinal layer, 18-day strobilated worms and 45-day adult worms was analyzed by immunofluorescence localization, and their secretory characteristics were analyzed preliminarily; in addition, quantitative real-time reverse transcription polymerase chain reaction was used to analyze their transcript levels in PSCs and 28-day strobilated worms stages. The phospholipid-binding activities of rEgAnxB3 and rEgAnxB38 were also analyzed. Results EgAnxB3 and EgAnxB38 are conserved and contain calcium-binding sites. Both rEgAnxB3 and rEgAnxB38 could be specifically recognized by the serum samples from E. granulosus-infected sheep, indicating that they had strong immunoreactivity. EgAnxB3 and EgAnxB38 were distributed in all stages of E. granulosus, and their transcript levels were high in the 28-day strobilated worms. They were found in liver tissues near the cysts. In addition, rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. Conclusions EgAnxB3 and EgAnxB38 contain calcium-binding sites, and rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. EgAnxB3 and EgAnxB38 were transcribed in PSCs and 28-day strobilated worms. They were expressed in all stages of E. granulosus, and distributed in the liver tissues near the hydatid cyst, indicating that they are secreted proteins that play a crucial role in the development of E. granulosus.

Molecular Characterization and Expression Analysis of Annexin B3 and B38 as Secretory Proteins in Echinococcus Granulosus

Background: Cystic Echinococcosis is a parasitic zoonotic disease, which poses a threat to public health and animal husbandry, and causes significant economic losses. Annexin is a kind of phospholipid binding protein with calcium ion binding activity, which has many functions.Methods: Two annexin protein family genes (EgAnxB3 and EgAnxB38) were cloned and molecular characterized using bioinformatic analysis. The immunoreactivity of rEgAnxB3 and rEgAnxB38 was investigated using western blotting. The distribution and transcript levels of EgAnxB3 and EgAnxB38 in different developmental stages of Echinococcus granulosus were analyzed by immunofluorescence localization and quantitative real-time reverse transcription PCR, and their secretory characteristics were analyzed preliminary. The phospholipid-binding activities of rEgAnxB3 and rEgAnxB38 were also tested.Results: EgAnxB3 and EgAnxB38 are conserved and contain calcium binding sites. Both rEgAnxB3 and rEgAnxB38 could be specifically recognized by the serum samples from E. granulosus-infected sheep, which have strong immunoreactivity. EgAnxB3 and EgAnxB38 were distributed in all stages of E. granulosus and had high transcript levels in the 28-day strobilated worms. They were found in liver tissues near the cysts. In addition, rEgAnxB3 had Ca2+-dependent phospholipid-binding properties.Conclusions: EgAnxB3 and EgAnxB38 contained calcium-binding sites, and rEgAnxB3 had Ca2+-dependent phospholipid-binding properties. rEgAnxB3 and rEgAnxB38 had strong immunoreactivity. EgAnxB3 and EgAnxB38 were transcribed in PSCs and worms. They were expressed in all stages of E. granulosus, and distributed in the liver tissues near the hydatid cyst, indicating that EgAnxB3 and EgAnxB38 are secreted proteins that play an crucial role in the development of E. granulosus.

System Approach for Building of Calcium-Binding Sites in Proteins

We introduce five new local metal cation (first of all, Ca2+) recognition units in proteins: Clampn,(n−2), Clampn,(n−1), Clampn,n, Clampn,(n+1) and Clampn,(n+2). In these units, the backbone oxygen atom of a residue in position “n” of an amino acid sequence and side-chain oxygen atom of a residue in position “n + i” (i = −2 to +2) directly interact with a metal cation. An analysis of the known “Ca2+-bound niches” in proteins has shown that a system approach based on the simultaneous use of the Clamp units and earlier proposed One-Residue (OR)/Three-Residue (TR) units significantly improves the results of constructing metal cation-binding sites in proteins.

Deciphering calcium-binding behaviors of casein phosphopeptides by experimental approaches and molecular simulation

Deciphering calcium-binding sites of CPPs and three calcium-binding modes with experimental methods and molecular simulation.