HSP90B1 Human, HEK

What is HSP90B1 and what are its fundamental functions in cellular processes?

HSP90B1 is an abundant molecular chaperone resident in the endoplasmic reticulum (ER) lumen and part of the Hsp90 family. It plays crucial roles in maintaining protein homeostasis in the secretory pathway by assisting in proper protein folding. HSP90B1 functions in signal transduction, protein folding, protein degradation, and morphologic evolution. It associates with numerous cochaperones and is involved in folding newly synthesized proteins or stabilizing and refolding denatured proteins after stress exposure .

Beyond protein folding, HSP90B1 participates in the intracellular trafficking of peptides from the extracellular space to the MHC class I antigen processing pathway of antigen presentation cells, highlighting its importance in immune function . Research has also revealed HSP90B1 is highly expressed in human gastric carcinoma BGC-823 cells throughout the cell cycle, suggesting potential roles in cellular proliferation .

How is recombinant HSP90B1 Human protein produced in HEK293 cells characterized?

HSP90B1 Human produced in HEK cells is characterized as a single, glycosylated polypeptide chain spanning from Asp22 to Glu798 and containing 789 amino acids, with a calculated molecular mass of 90.9kDa. The recombinant protein typically includes a 2 amino acid N-terminal linker, a 4 amino acid C-terminal linker, and a 6 amino acid His tag at the C-terminus to facilitate purification .

For experimental preparation, the protein is supplied as a filtered white lyophilized (freeze-dried) powder. The formulation consists of HSP90B1 in phosphate-buffered saline (PBS) at pH 8.0, supplemented with stabilizers including 1% (w/v) sucrose and 4% (w/v) mannitol . This formulation enhances stability during storage and reconstitution.

What are the optimal handling methods for HSP90B1 recombinant protein in laboratory settings?

For researchers working with HSP90B1 recombinant protein, following these methodological guidelines ensures optimal results:

• Storage: Store lyophilized protein at -20°C to maintain long-term stability.

• Reconstitution: Add deionized water to prepare a working stock solution of approximately 0.5mg/ml and allow the lyophilized pellet to dissolve completely.

• Aliquoting: After reconstitution, aliquot the product to avoid repeated freezing/thawing cycles that can compromise protein integrity .

• Sterility considerations: The recombinant HSP90B1 is not provided as a sterile product. For cell culture applications, filter through an appropriate sterile filter (0.2μm or 0.4μm) before use .

• Solubility verification: After reconstitution, visual inspection should confirm complete dissolution without precipitates.

These handling procedures maintain protein activity and ensure experimental reproducibility when working with HSP90B1 recombinant preparations.

How does HSP90B1 interact with Toll-like receptors (TLRs) and what are the functional implications?

HSP90B1 serves as a master chaperone for multiple Toll-like receptors (TLRs) including TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9 . This chaperoning relationship is critical for proper TLR folding, maturation, and function. Methodologically, this interaction has been verified through:

• Conditional knockout models: B cell-specific HSP90B1-deficient mice show attenuated antibody production specifically in response to TLR stimulation, while maintaining normal B-cell numbers and immunoglobulin expression .

• Association studies: Genetic variants in HSP90B1 (rs10507172, rs10507173, and rs1920413) have been associated with altered BCG-induced IL-2 secretion (p=0.017 for rs10507172 and p=0.03 for rs10507173 and rs1920413) .

• Functional assays: HSP90B1 ablation impairs TLR-mediated inflammatory responses in myeloid cells, demonstrating its necessity for proper TLR signaling .

The functional implications extend to both innate and adaptive immunity, particularly in host defense against pathogens like Mycobacterium tuberculosis, where HSP90B1 genetic variants have been associated with susceptibility to TB disease in case-control studies .

What experimental models are available for studying HSP90B1 function?

Researchers investigating HSP90B1 can utilize several experimental model systems:

• Conditional knockout mouse models: B cell-specific HSP90B1-deficient mice generated using Cre-loxP technology allow for tissue-specific analysis of HSP90B1 function. These models have revealed that HSP90B1 is essential for optimal B-cell responses to TLR stimulation but dispensable for B-cell survival .

• CRISPR/Cas9 knockout cell lines: HEK293T cells with HSP90B1 knocked out (10 bp deletion in exon 1) provide a clean genetic background for studying protein function in vitro . These models have been validated by Western blot and are commercially available with matched wild-type controls.

• Pharmacological inhibition systems: HSP90B1 can be inhibited using specific inhibitors like PU-WS13, which allows for temporal control of inhibition and dose-response studies . Additional inhibitors include geldanamycin (GA), PU-H71, and novobiocin, though these target the broader Hsp90 family .

• Domain-specific mutants: Recombinant HSP90B1 with mutations in functional domains, such as the TPR1 mutant K8A (unable to bind Hsp70) and TPR2A mutant K229A (unable to bind Hsp90), enable detailed study of domain-specific functions .

Each model system offers distinct advantages for investigating different aspects of HSP90B1 biology, from molecular interactions to physiological functions.

What role does HSP90B1 play in B-cell immune responses?

HSP90B1's role in B-cell function has been clarified through targeted genetic deletion studies. Contrary to earlier hypotheses suggesting a role in immunoglobulin assembly, HSP90B1 primarily optimizes B-cell function by chaperoning Toll-like receptors (TLRs) and integrins, not immunoglobulins .

Key experimental findings include:

• Conditional knockout studies: B cell-specific HSP90B1-deficient mice show normal B-cell numbers and normal expression levels of B220, IgM, and IgD, demonstrating that BCR assembly remains intact without HSP90B1 .

• Functional deficits: The primary defect in HSP90B1-null B cells is attenuated antibody production specifically in response to TLR stimulation .

• Subpopulation effects: HSP90B1 deficiency leads to significant reductions in splenic marginal zone B cells (B220+CD21+CD23-) and peritoneal B1 cells (B220+IgM+CD5+) . Interestingly, these cell populations were increased in the blood of knockout mice, suggesting HSP90B1 may be involved in proper trafficking or homing rather than development.

These findings resolve the long-standing question about HSP90B1 in B-cell biology, demonstrating its specific role in optimizing TLR-mediated responses rather than directly affecting immunoglobulin production or B-cell survival.

How does the HSP90B1-Hsp70-Hop ternary complex influence proteasomal activity?

The HSP90B1-Hsp70-Hop ternary complex plays a critical role in regulating proteasomal activity through a mechanistically complex relationship. Research using multiple experimental approaches has revealed:

• Knockout effects: Cells lacking the co-chaperone Hop (STIP1/STI1) show reduced steady-state proteasomal activity. This finding is conserved in both mammalian and yeast models .

• Domain-specific mutations: Hop mutants that cannot form the ternary complex with Hsp70 and Hsp90 (K8A, K229A, and double mutants) behave like complete loss-of-function mutants regarding proteasomal regulation, demonstrating that the intact ternary complex is required .

• Pharmacological inhibition: Inhibition of either Hsp70 (with JG-98) or Hsp90 (with GA) leads to reduced proteasomal activity. Interestingly, combined suboptimal inhibition does not show additive or synergistic effects, suggesting they function in the same pathway .

• HSP90 isoform deletion: Deletion of genes encoding either Hsp90α (HSP90AA1) or Hsp90β (HSP90AB1) in HEK293T cells results in reduced steady-state proteasomal activity, indicating both isoforms contribute to this regulation .

This complex influences steady-state proteasomal activity rather than the rate of proteasomal activity, suggesting a role in maintaining basal proteasome function rather than acutely regulating degradation rate .

What is the significance of HSP90B1 in cancer biology and how might it be targeted therapeutically?

HSP90B1 has emerged as a promising target for cancer diagnosis, prognosis, and therapy based on comprehensive multi-omics analyses and experimental validations. Key findings include:

• Expression patterns: HSP90B1 is highly expressed in various tumors and often correlates with poor prognosis . Single-cell RNA sequencing analysis has revealed significantly higher expression in tumor cells compared to surrounding normal cells, providing a potential therapeutic window .

• Immune associations: HSP90B1 expression negatively correlates with CD8+ T cells in most tumors, suggesting an immunosuppressive role. It also positively correlates with microsatellite instability, tumor mutational burden, and expression of immune checkpoint genes .

• Pathway connections: HSP90B1 expression positively correlates with tumor metabolism and cell cycle-related pathways. It shows negative correlation with immunostimulatory genes and positive correlation with immunosuppressive genes .

• Therapeutic potential: The HSP90B1 inhibitor PU-WS13 significantly suppresses cancer cell proliferation in both leukemic and solid tumor models. Importantly, it also reduces expression of the cancer cell surface immune checkpoint PD-L1, potentially enhancing anti-tumor immunity .

These findings suggest HSP90B1 inhibition could offer dual therapeutic benefits: direct anti-tumor effects and enhancement of anti-tumor immunity by reducing immunosuppressive mechanisms.

How can researchers resolve conflicting data on HSP90B1 function across different experimental systems?

Researchers face several challenges when interpreting sometimes conflicting data on HSP90B1 function. Methodological approaches to resolve these conflicts include:

By employing these methodological approaches, researchers can develop more nuanced and accurate models of HSP90B1 function that account for its diverse roles across cellular contexts.