Recombinant Pongo abelii McKusick-Kaufman/Bardet-Biedl syndromes putative chaperonin (MKKS), partial

What is MKKS and what are its primary functions in cellular processes?

MKKS (McKusick-Kaufman/Bardet-Biedl syndromes putative chaperonin), also known as BBS6, functions as a molecular chaperone that assists in protein folding through ATP hydrolysis. The protein plays several critical roles in cellular processes:

• Facilitates proper folding of client proteins

• Contributes to BBSome assembly, a complex involved in ciliogenesis

• Regulates transport vesicles to the cilia

• Participates in protein processing for limb, cardiac, and reproductive system development

• May play a role in cytokinesis

As a member of the TCP-1 chaperonin family, MKKS represents an important component of cellular protein quality control systems. Its dysfunction is associated with developmental abnormalities characteristic of Bardet-Biedl Syndrome.

How is MKKS related to Bardet-Biedl Syndrome pathogenesis?

MKKS (also designated as BBS6) plays a central role in Bardet-Biedl Syndrome (BBS) pathogenesis through several mechanisms:

• It is one of three genes (along with BBS10 and BBS12) encoding chaperonin-like proteins involved in BBS

• Mutations in these three chaperonin-like genes account for approximately 50% of clinically-diagnosed BBS cases, highlighting their critical importance

• BBS is classified as a ciliopathy, with defects in cilia structure and/or function causing the syndrome's characteristic features

• MKKS localizes to centrosomes and ciliary basal bodies, supporting its role in ciliogenesis

• The syndrome's six diagnostic features (retinal dystrophy, obesity, polydactyly, cognitive impairment, and renal and urogenital anomalies) reflect the diverse tissues affected by MKKS dysfunction

The classification of BBS as a chaperonopathy (disease caused by chaperone defects) underscores the importance of protein folding and quality control in maintaining ciliary function across multiple tissue types.

What are the optimal methods for expressing and purifying recombinant MKKS protein?

For successful expression and purification of recombinant Pongo abelii MKKS, researchers should consider the following methodological approach:

Expression Systems Selection:

• Insect cell expression system: Based on successful approaches with related proteins, Sf9 cells with baculovirus vectors offer advantages for chaperonin protein expression

• Mammalian expression systems: HEK293 or CHO cells may provide more physiologically relevant post-translational modifications

• E. coli systems: While less optimal for complex eukaryotic proteins, they can be used with solubility-enhancing tags

Expression Strategy:

• Generate a construct with an appropriate affinity tag (His6 is commonly used)

• Include a protease cleavage site for tag removal if needed for functional studies

• Consider codon optimization for the expression host

• Optimize expression conditions (temperature, induction time, media composition)

Purification Protocol:

• Cell lysis: Use gentle methods to preserve protein structure (e.g., freeze-thaw, mild detergents)

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Intermediate purification: Ion exchange chromatography based on MKKS theoretical pI

• Polishing step: Size exclusion chromatography to isolate monomeric or properly assembled MKKS

Quality Control Assessment:

• Purity analysis by SDS-PAGE

• Western blot to confirm identity

• Mass spectrometry for intact mass verification

• Circular dichroism to assess secondary structure

• Dynamic light scattering to evaluate size distribution and aggregation state

For researchers requiring active MKKS for functional studies, special attention must be given to preserving the native conformation throughout the purification process.

How can researchers assess MKKS chaperone function in vitro?

Evaluating the chaperone function of recombinant MKKS requires multiple complementary approaches:

ATP Hydrolysis Assays:

• Measure ATPase activity using malachite green phosphate detection or NADH-coupled enzymatic assays

• Compare basal vs. substrate-stimulated ATP hydrolysis rates

• Analyze how mutations affect ATP hydrolysis kinetics

• Determine Michaelis constants for ATP hydrolysis

Protein Folding Assistance Assays:

• Monitor prevention of aggregation of model substrate proteins under stress conditions

• Assess refolding of chemically denatured substrates

• Measure thermal stabilization of client proteins

• Quantify protection of enzymatic activity of substrate proteins

BBSome Assembly Evaluation:

• In vitro reconstitution of BBSome components with and without MKKS

• Analysis of complex formation using size exclusion chromatography coupled with multi-angle light scattering

• Assessment of BBSome assembly kinetics using fluorescently labeled components

• Comparison of wild-type vs. mutant MKKS in promoting proper assembly

Structural Studies:

• Characterize MKKS-substrate interactions using hydrogen-deuterium exchange mass spectrometry

• Analyze conformational changes upon nucleotide binding using fluorescence spectroscopy

• Visualize chaperone-substrate complexes using cryo-electron microscopy

The combination of these approaches provides a comprehensive assessment of MKKS chaperone function and how it might be impaired in disease-causing variants.

What animal and cellular models are most effective for studying MKKS function?

Several model systems have proven valuable for investigating MKKS function in vivo:

Mouse Models:

• Mkksko/ko (MKKS knockout): Exhibits phenotypes reminiscent of BBS, including retinal degeneration

• Cep290rd16/rd16;Mkksko/ko (double knockout): Shows variable phenotypes ranging from near-normal to significant retinal abnormalities, providing insights into genetic interactions

• Conditional tissue-specific knockouts: Allow investigation of MKKS function in specific tissues without developmental complications

Cellular Models:

• Primary ciliated cells: Derived from patients with MKKS mutations or from animal models

• CRISPR-engineered cell lines: Created with specific MKKS variants to study their effects on ciliogenesis

• iPSC-derived organoids: Provide three-dimensional tissue context for studying ciliary function

Experimental Approaches with Models:

• Immunofluorescence microscopy to assess ciliary structure and BBSome localization

• Live cell imaging to track protein dynamics in cilia

• Electron microscopy to examine ultrastructural ciliary defects

• Transcriptomic and proteomic analyses to identify downstream effects of MKKS dysfunction

• Rescue experiments to confirm specificity of observed phenotypes

When selecting a model system, researchers should consider the specific aspect of MKKS function they wish to study and choose the model that best recapitulates the relevant human pathophysiology.

How do specific mutations in MKKS affect protein folding and chaperone function?

The impact of mutations on MKKS function involves complex molecular mechanisms that can be analyzed through multiple approaches:

Mutation Classification and Effects:

One example from the literature is the p.A6T variant, which changes a conserved hydrophobic alanine to a hydrophilic threonine residue. This alanine is conserved across mammals, suggesting functional importance . While blosum62 scoring suggested this change might be pathological, other prediction tools (SIFT and Polyphen) yielded different results, highlighting the complexity of variant interpretation .

Experimental Approaches to Assess Mutational Impact:

• Thermal stability assessments using differential scanning fluorimetry

• Structural analysis of mutant proteins using crystallography or cryo-EM

• Molecular dynamics simulations to predict conformational changes

• Domain mapping to identify critical functional regions

• Interaction studies to determine effects on protein-protein binding

What is the precise role of MKKS in BBSome assembly and ciliogenesis?

MKKS plays a specialized role in BBSome assembly and subsequent ciliogenesis through several mechanisms:

• Functions as part of a chaperonin complex with BBS10 and BBS12 to assist in the folding of BBSome components

• Localizes to centrosomes and ciliary basal bodies, positioning it ideally for BBSome assembly

• Facilitates the proper assembly of the BBSome, an octameric protein complex comprising BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBIP10

• Influences the regulation of vesicle transport to the cilia, a critical process for ciliary membrane composition

• May coordinate with other chaperone systems to ensure proper folding of ciliary proteins

The BBSome functions as a coat complex that sorts membrane proteins to primary cilia. MKKS dysfunction disrupts this process, leading to ciliary defects that manifest as BBS symptoms across multiple organ systems.

Current research questions focus on:

• The temporal sequence of MKKS action during BBSome assembly

• How MKKS cooperates with BBS10 and BBS12 in the chaperone complex

• The specific client proteins that require MKKS for proper folding

• The mechanisms by which MKKS influences vesicular trafficking to cilia

How do genotype-phenotype correlations inform our understanding of MKKS-related disorders?

Genotype-phenotype correlations in MKKS-related disorders reveal complex relationships between specific genetic variants and clinical manifestations:

Clinical Phenotype Variability:
The literature describes patients with MKKS variants presenting with diverse phenotypes. For instance:

• Patient MOGL 767 (heterozygous p.A6T variant): Presented with lifelong nyctalopia, color vision loss, and rapidly progressive retinal dystrophy, with visual acuity deteriorating from 20/80 to light perception within one year

• Patient MOGL 1110: Presented with lifelong nyctalopia, high myopia, non-detectable ERGs, and progressive vision loss from age 7 to 17 years

Factors Influencing Phenotypic Expression:

Research Approaches to Improve Correlations:

• Deep phenotyping using standardized assessment protocols

• Comprehensive genetic analysis including potential modifier genes

• Functional characterization of variants using in vitro and in vivo models

• Long-term natural history studies to track phenotypic progression

• Creation of variant databases with detailed clinical information

These correlations are essential for prognosis, genetic counseling, and targeted therapeutic development.

What insights can comparative analysis of MKKS across species provide about its evolution?

Comparative analysis of MKKS across species reveals important evolutionary patterns and functional constraints:

Conservation Patterns:

• Key functional residues, such as Ala6, show conservation across all mammals, indicating selective pressure

• ATP-binding and hydrolysis domains typically display the highest conservation

• The chaperonin domain architecture is preserved while allowing for species-specific adaptations

• MKKS, BBS10, and BBS12 form a distinct evolutionary group that diverged from canonical Group II chaperonins

Evolutionary Significance:
The emergence and conservation of MKKS and other BBS-related chaperonins correlate with the evolution of complex ciliary structures in eukaryotes. This evolutionary history provides insights into:

• The timing of functional specialization of chaperonins for ciliary protein folding

• Co-evolution of the BBSome complex with its assembly chaperones

• The development of tissue-specific functions of cilia across vertebrate evolution

• The emergence of vulnerabilities that lead to ciliopathies like BBS

Methodological Approaches for Evolutionary Analysis:

• Multiple sequence alignment of MKKS orthologs across diverse species

• Phylogenetic analysis to determine evolutionary relationships

• Calculation of selection pressures (dN/dS ratios) to identify conserved functional domains

• Ancestral sequence reconstruction to infer evolutionary transitions

• Structure-based analysis to correlate sequence conservation with functional domains

How does the structure and function of Pongo abelii MKKS compare to human MKKS?

The comparison between Pongo abelii (Sumatran orangutan) and human MKKS provides valuable insights into primate-specific adaptations of this important chaperonin:

Sequence Comparison:
While the search results don't provide a direct comparison, primate orthologs typically share high sequence identity (>95%) in conserved proteins like chaperonins. Key features likely include:

• Nearly identical ATP-binding and hydrolysis domains

• Highly conserved substrate recognition regions

• Potential differences in regulatory regions or species-specific interaction sites

Functional Implications:

• The high conservation suggests similar roles in BBSome assembly and ciliogenesis

• Any differences may reflect species-specific adaptations in ciliary function

• Studying the orangutan protein may reveal which features are universally required versus those that are primate-specific

Research Applications:

• Pongo abelii MKKS can serve as a model for human MKKS in structural and functional studies

• Comparative analysis may identify critical versus dispensable regions for therapeutic targeting

• Understanding primate-specific features could help interpret human variants of uncertain significance

Researchers working with the recombinant Pongo abelii MKKS should be aware of potential subtle differences from human MKKS that might affect interactions with other proteins, particularly those that might be less conserved across primate species.

How can MKKS research inform therapeutic approaches for Bardet-Biedl Syndrome?

Research on MKKS provides several avenues for therapeutic development in Bardet-Biedl Syndrome:

Mechanism-Based Therapeutic Strategies:

Target Tissues for Intervention:

• Retina: To address progressive vision loss

• Hypothalamus: To manage obesity and metabolic dysfunction

• Kidneys: To prevent or treat renal abnormalities

• Reproductive system: To address fertility issues

Research-to-Clinic Translation Challenges:

• Genetic heterogeneity of BBS necessitates personalized approaches

• Multiple affected tissues require multi-system therapeutic strategies

• Timing of intervention is critical, especially for developmental features

• Delivery methods must be optimized for each target tissue

• Long-term safety and efficacy assessment is essential

Understanding the precise molecular and cellular effects of different MKKS mutations is crucial for selecting appropriate therapeutic approaches for individual patients.

What diagnostic applications can be developed based on MKKS research?

MKKS research enables the development of several diagnostic approaches for BBS and related ciliopathies:

Molecular Diagnostic Methods:

• Targeted gene panel testing focusing on MKKS and other BBS genes

• Development of functional assays to assess the pathogenicity of MKKS variants

• RNA-based testing to detect splice variants that may be missed by DNA sequencing

• Copy number variation analysis to identify large deletions or duplications

Biomarker Development:

• Identification of protein or metabolic signatures associated with MKKS dysfunction

• Development of assays to measure BBSome assembly efficiency as a functional readout

• Ciliary morphology and function assessment in accessible patient cells

Clinical Implementation Strategies:

• Integration of molecular and clinical findings for improved diagnostic accuracy

• Development of diagnostic algorithms incorporating MKKS testing at appropriate stages

• Implementation of newborn screening approaches for early identification

• Design of predictive testing protocols for at-risk family members

Benefits of Advanced Diagnostics:

• Earlier diagnosis enabling timely intervention

• More precise prognosis based on specific molecular defects

• Facilitation of genetic counseling for affected families

• Identification of patients suitable for targeted therapies

• Creation of well-characterized cohorts for clinical trials

The continued characterization of MKKS variants and their functional consequences will progressively enhance diagnostic accuracy and guide appropriate clinical management.