Recombinant Macaca mulatta Heat shock protein beta-8 (HSPB8)

What is the basic structure of Macaca mulatta HSPB8 and how does it compare to human HSPB8?

Macaca mulatta HSPB8, like its human counterpart, is a small heat shock protein of approximately 22 kDa. The protein consists of approximately 196 amino acids with a conserved alpha-crystalline domain (ACD) located between amino acids 86-176. This domain is crucial for protein-protein interactions, while the N-terminal and C-terminal regions facilitate oligomerization. The high sequence homology between primate HSPB8 proteins (human HSPB8 shares 94% amino acid identity with both mouse and canine versions) suggests that Macaca mulatta HSPB8 likely maintains similar structural features to the human protein .

Unlike other members of the HSPB family (HSPB1, HSPB2, HSPB4, and HSPB5), HSPB8 does not contain the conserved I/V-X-I/V motif responsible for oligomeric assembly. Instead, HSPB8 contains a hydrophobic pocket that mediates binding to the IPV domains present in the BAG3 protein, explaining its preferential binding to BAG3 rather than forming large homo-oligomers .

What are the primary cellular functions of HSPB8 relevant to research applications?

HSPB8 serves multiple critical cellular functions that make it valuable for diverse research applications:

• Protein Quality Control: HSPB8 is a key component of the chaperone-assisted selective autophagy (CASA) complex, which promotes the selective degradation of misfolded proteins to counteract cellular stress . The CASA complex comprises molecular chaperones HSPA8 and HSPB8, along with co-chaperones BAG3 and STUB1 .

• Cell Protection: As a heat shock protein, HSPB8 helps protect cells under stress from factors including infection, inflammation, toxin exposure, elevated temperature, injury, and disease. It specifically blocks signals that lead to cellular apoptosis .

• Cytoskeletal Regulation: HSPB8 is involved in cell movement and stabilizing the cellular cytoskeleton .

• Protein Homeostasis: It assists in folding newly produced proteins and refolding damaged proteins throughout the body .

• Cell Division Regulation: HSPB8 participates in cell division machinery, regulating chromosome segregation and cell cycle arrest in the G0/G1 phase .

• Inflammatory Response: The protein regulates dendritic cell maturation and cytokine production as part of inflammatory processes .

For research applications, these functions make HSPB8 particularly valuable in studying neurodegenerative diseases, cancer biology, and fundamental cellular stress response mechanisms.

Which expression systems are optimal for producing functional recombinant Macaca mulatta HSPB8?

For producing functional recombinant Macaca mulatta HSPB8, several expression systems can be employed, with E. coli being the most commonly used for small heat shock proteins. Based on available research data and protocols for similar proteins:

E. coli Expression System (Recommended):

• Advantages: High yield, cost-effective, rapid production

• Vector recommendations: pET series vectors with T7 promoter systems

• Strain recommendations: BL21(DE3), Rosetta(DE3), or Origami for proteins requiring disulfide bonds

• Expression conditions: Induction with 0.5-1.0 mM IPTG at 18-25°C for 16-18 hours can reduce inclusion body formation and improve solubility

The presence of an E. coli-derived recombinant human HSPB8 in the research literature suggests that prokaryotic expression systems are effective for producing functional HSPB8 . When expressing HSPB8, consider that:

• Lower induction temperatures (16-20°C) may enhance proper folding and solubility

• Co-expression with chaperones may improve folding and solubility

• Fusion tags (His, GST, MBP) can facilitate purification and may enhance solubility

For specialized applications requiring post-translational modifications:

• Mammalian expression systems (HEK293, CHO cells) may be necessary

• Insect cell systems (Sf9, Hi5) using baculovirus expression vectors can provide intermediate complexity post-translational modifications

What are the most effective purification strategies for recombinant HSPB8, and how can researchers validate protein quality?

Purification Strategy:

• Affinity Chromatography:

  • His-tagged HSPB8: Ni-NTA or IMAC purification

  • GST-tagged HSPB8: Glutathione Sepharose purification

  • Typical binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole for His-tagged proteins

• Size Exclusion Chromatography (SEC):

  • Essential second step to remove aggregates and ensure homogeneity

  • Recommended columns: Superdex 75 or Superdex 200 for HSPB8 depending on oligomeric state

• Ion Exchange Chromatography:

  • Can be used as an intermediate or polishing step

  • Given the predicted pI of HSPB8, anion exchange (Q-Sepharose) at pH 8.0 is often suitable

Quality Validation Methods:

• SDS-PAGE and Western Blotting:

  • Confirm molecular weight (approximately 22 kDa)

  • Commercially available antibodies can detect HSPB8 across human, mouse, and rat species, suggesting cross-reactivity with Macaca mulatta HSPB8

• Mass Spectrometry:

  • Validate exact molecular weight and sequence coverage

  • Identify potential post-translational modifications

• Dynamic Light Scattering (DLS):

  • Assess homogeneity and oligomeric state

  • Monitor for unwanted aggregation

• Functional Assays:

  • Chaperone activity assays using model substrates

  • BAG3 binding assays to confirm proper folding of the BAG3 interaction domain

• Circular Dichroism (CD):

  • Evaluate secondary structure integrity

  • Thermal stability assessment

When working with recombinant HSPB8, researchers should store the purified protein according to standard protocols: avoid repeated freeze-thaw cycles, store at -70°C for long-term storage, and maintain at 2-8°C under sterile conditions after reconstitution for up to 1 month .

How can recombinant Macaca mulatta HSPB8 be utilized in studying neurodegenerative disease mechanisms?

Recombinant Macaca mulatta HSPB8 serves as a valuable tool for investigating neurodegenerative disease mechanisms, particularly those involving protein misfolding and aggregation. Several methodological approaches can be implemented:

• In vitro Aggregation Models:

  • Recombinant HSPB8 can be used to study its chaperone activity against disease-relevant protein aggregates (e.g., SOD1, TDP-43, or mutant proteins associated with CMT2L)

  • Protocol: Incubate fluorescently labeled aggregation-prone proteins with varying concentrations of recombinant HSPB8 and monitor aggregation kinetics using thioflavin T fluorescence or light scattering

• Cell-Based Models of Protein Aggregation:

  • Transfect neuronal cell lines with disease-relevant proteins and assess how co-expression or treatment with recombinant HSPB8 modifies aggregate formation and clearance

  • Quantify using immunofluorescence microscopy or biochemical fractionation methods

• Studying HSPB8 Mutations:

  • Generate disease-associated mutations (K141E, P173SfsX43) in recombinant Macaca mulatta HSPB8 to study their effects on:

    • Protein structure and stability

    • Interaction with CASA complex components

    • Chaperone activity

  • These mutations have been linked to Charcot-Marie-Tooth type 2L, distal hereditary motor neuropathy type IIa, and distal myopathy with rimmed vacuoles

• CASA Complex Reconstitution:

  • Assemble in vitro CASA complexes using recombinant HSPB8, BAG3, HSPA8, and STUB1

  • Assess how disease mutations affect complex formation and function

Recent research has expanded our understanding of HSPB8-associated diseases. Initially linked only to Charcot-Marie-Tooth type 2L and distal hereditary motor neuropathy type IIa, mutations in HSPB8 are now known to cause myopathies with histologic features of myofibrillar myopathy with aggregates and rimmed vacuoles . The recently identified c.515delC mutation appears to be a hotspot and causes a more severe phenotype in males with earlier onset of myopathy .

What experimental approaches can be used to investigate HSPB8's role in the CASA complex in neuronal models?

The CASA complex represents a critical protein quality control mechanism in neurons. To investigate HSPB8's role within this complex in neuronal models, researchers can employ several methodological approaches:

• Co-Immunoprecipitation Studies:

  • Use recombinant HSPB8 as bait to identify interacting partners in neuronal lysates

  • Compare wild-type vs. mutant HSPB8 to determine how disease mutations affect complex formation

  • Protocol: Immobilize recombinant HSPB8 on appropriate beads, incubate with neuronal lysates, wash, and identify bound proteins by Western blot or mass spectrometry

• Proximity Ligation Assays (PLA):

  • Visualize endogenous protein-protein interactions between HSPB8 and other CASA components in intact neurons

  • Advantages: Single-molecule resolution, spatial information about interaction sites within neurons

• CRISPR/Cas9-Mediated Genome Editing:

  • Generate HSPB8 knockout or knock-in neuronal cell lines or primary cultures

  • Assess consequences on:

    • Autophagy flux (using LC3-II/I ratio and p62 levels)

    • Protein aggregation (using proteinopathy models)

    • Neuronal survival under stress conditions

• Fluorescence Resonance Energy Transfer (FRET):

  • Create fluorescently tagged HSPB8 and CASA components to monitor real-time interactions in living neurons

  • Quantify interaction dynamics under normal and stress conditions

• In Vitro Reconstitution Assays:

  • Combine purified recombinant HSPB8, BAG3, HSPA8, and STUB1 to reconstitute the CASA complex

  • Assess client protein ubiquitination and degradation rates

Research has established that HSPB8 works with the CASA complex to maintain protein quality control, particularly in mechanically strained tissues such as skeletal muscle, heart, and lung . The role in neurons specifically involves protecting against misfolded proteins that can lead to neurodegeneration.

What are the key considerations when designing experiments to study phosphorylation-dependent regulation of HSPB8 function?

Phosphorylation is known to regulate HSPB8 function, with phosphorylation at Ser57 specifically blocking HSPB8 chaperone activity . When designing experiments to investigate phosphorylation-dependent regulation of recombinant Macaca mulatta HSPB8, researchers should consider:

How can researchers effectively study species-specific differences between human and Macaca mulatta HSPB8 for translational research applications?

Understanding species-specific differences between human and Macaca mulatta HSPB8 is crucial for translational research. Here are methodological approaches to characterize these differences:

• Comparative Sequence Analysis:

  • Perform comprehensive sequence alignment of human and Macaca mulatta HSPB8

  • Identify non-conserved residues, with special attention to:

    • The alpha-crystalline domain (aa 86-176)

    • Known functional regions (e.g., BAG3 interaction sites)

    • Disease-associated mutation sites (e.g., K141, position 173)

• Structural Comparison:

  • Determine crystal or solution structures of both species' HSPB8 using X-ray crystallography or NMR

  • Generate homology models if experimental structures are unavailable

  • Compare surface charge distribution, hydrophobic patches, and potential binding interfaces

• Functional Comparison Assays:

  • Chaperone activity: Compare thermal aggregation prevention efficiency using model substrates

  • Client specificity: Identify differences in substrate preference using protein arrays

  • Binding partner affinities: Measure interaction strength with key partners like BAG3 using surface plasmon resonance

• Cell-Based Cross-Species Complementation:

  • Perform rescue experiments in HSPB8-depleted human cells using either human or Macaca mulatta HSPB8

  • Assess capacity to:

    • Prevent protein aggregation

    • Maintain autophagy flux

    • Protect against stress-induced cell death

• Interactome Analysis:

  • Perform pull-down experiments using recombinant human and Macaca mulatta HSPB8 with cell lysates from both species

  • Identify species-specific interaction partners by mass spectrometry

  • Validate key differences using co-immunoprecipitation or proximity ligation assays

Researchers should note that human HSPB8 shares high sequence homology with other mammals (94% amino acid identity with both mouse and canine HSPB8) , suggesting that Macaca mulatta HSPB8 likely maintains similar structural and functional properties to human HSPB8, making it valuable for translational research.

What methods are most effective for studying HSPB8 interactions with other components of the proteostasis network?

Studying HSPB8 interactions with other components of the proteostasis network requires multiple complementary approaches:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Use tagged recombinant Macaca mulatta HSPB8 as bait

  • Protocol:

    • Express tagged HSPB8 in relevant cell types

    • Perform gentle lysis to preserve protein complexes

    • Capture HSPB8 and associated proteins using appropriate affinity matrix

    • Identify interacting partners by LC-MS/MS

  • Variations: SILAC or TMT labeling for quantitative comparison between conditions

• Bioluminescence Resonance Energy Transfer (BRET) or FRET:

  • Create fusion proteins of HSPB8 and potential interactors with compatible BRET/FRET pairs

  • Measure energy transfer as an indication of protein proximity (<10 nm)

  • Advantages: Can be performed in living cells, allows real-time monitoring of dynamic interactions

• Yeast Two-Hybrid Screening:

  • Use HSPB8 as bait to screen for novel interactors from cDNA libraries

  • Follow with validation using orthogonal methods

  • Consider split-ubiquitin system for membrane-associated interactors

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Quantitatively measure binding kinetics and affinities between purified HSPB8 and partners

  • Determine kon, koff, and KD values

  • Compare wild-type vs. mutant HSPB8 binding properties

• Protein Complementation Assays:

  • Split reporter systems (luciferase, GFP, β-lactamase)

  • Particularly useful for detecting interactions in specific cellular compartments

The CASA complex, of which HSPB8 is a critical component, comprises the molecular chaperones HSPA8 and HSPB8 and the co-chaperones BAG3 and STUB1 . Research has shown that two molecules of HSPB8 complement one BAG3 molecule, forming a functional chaperone complex . This stoichiometry should be considered when designing interaction studies.

How can advanced imaging techniques be employed to visualize HSPB8 dynamics in neuronal and muscle cells?

Advanced imaging techniques provide powerful tools for visualizing HSPB8 dynamics in cellular contexts relevant to disease states:

• Super-Resolution Microscopy Approaches:

  • STED (Stimulated Emission Depletion) Microscopy:

    • Resolution: ~50 nm

    • Applications: Visualizing HSPB8 distribution relative to cytoskeletal elements and stress granules

  • PALM/STORM (Photoactivated Localization/Stochastic Optical Reconstruction Microscopy):

    • Resolution: ~20 nm

    • Applications: Single-molecule tracking of HSPB8 in living cells, quantifying oligomerization states

  • SIM (Structured Illumination Microscopy):

    • Resolution: ~100 nm

    • Applications: Live-cell imaging of HSPB8 redistribution during stress response

• Live-Cell Imaging of HSPB8 Dynamics:

  • FRAP (Fluorescence Recovery After Photobleaching):

    • Measure mobility and binding dynamics of HSPB8-GFP fusion proteins

    • Compare mobility in different cellular compartments and under stress conditions

  • Photo-convertible Fluorescent Protein Fusions:

    • Track movement of specific HSPB8 pools within cells

    • Protocol: Express HSPB8 fused to Dendra2 or mEos, photoconvert a specific region, and track movement

• Multi-color Imaging of HSPB8 with CASA Components:

  • Simultaneous visualization of HSPB8 with BAG3, HSPA8, and autophagic markers

  • Correlate spatial relationships with functional outcomes

  • Appropriate for studying pathological conditions with protein aggregates and rimmed vacuoles

• Correlative Light and Electron Microscopy (CLEM):

  • Combine fluorescence identification of HSPB8-positive structures with ultrastructural characterization

  • Particularly useful for studying HSPB8 association with:

    • Rimmed vacuoles in myopathies

    • Myofibrillar aggregates

    • Autophagic structures

• Biosensor Applications:

  • HSPB8 fusion with conformational stress sensors

  • Monitor chaperone activity in real time within living cells

When studying neuromuscular diseases, these imaging approaches can reveal how HSPB8 localizes relative to pathological features such as the myofibrillar aggregates and rimmed vacuoles observed in patients with HSPB8 mutations .