Recombinant Human Kelch-like protein 35 (KLHL35)

What is the basic structural organization of human KLHL35?

Human KLHL35 belongs to the evolutionarily conserved Kelch protein superfamily, which contains 63 alternate protein coding members across three subfamilies: Kelch-like (KLHL), Kelch-repeat and BTB domain containing (KBTBD), and Kelch domain containing (KLHDC) proteins . As a member of the KLHL subfamily, KLHL35 likely features the characteristic BTB/POZ (Broad Complex, Tramtrack, and Bric-a-brac/Poxvirus and Zinc finger) domain at its N-terminus, followed by a BACK (BTB and C-terminal Kelch) domain and multiple kelch repeats that form a β-propeller structure at the C-terminus. The BTB domain typically facilitates protein dimerization, while the kelch repeats mediate protein-protein interactions with various binding partners, similar to other characterized kelch-like proteins .

How do the kelch repeats in KLHL35 contribute to its function?

The kelch repeats in KLHL35, like those in other Kelch family proteins, likely form a β-propeller structure that creates a protein-protein interaction platform. Each kelch repeat consists of 40-50 amino acids with highly conserved sequences, collectively forming a specialized binding surface . In related proteins like KLEIP, these kelch repeats have been demonstrated to interact with filamentous actin (F-actin), suggesting potential cytoskeletal interaction capabilities . For researchers investigating KLHL35 function, it's important to analyze the conservation pattern across the kelch repeats, as studies on other family members have shown varying degrees of conservation between different repeats, with some (like the fourth domain in PPKL) showing higher identity between species than others . Understanding these patterns can help predict potential binding partners and functional domains for targeted mutation studies.

What post-translational modifications are known to regulate KLHL35 activity?

While specific post-translational modifications of KLHL35 are not extensively characterized in the provided research data, studies on related Kelch family proteins suggest several possible regulatory mechanisms that researchers should investigate. Phosphorylation is a particularly important consideration, as demonstrated by the functional importance of phosphatase domains in related proteins like PPKL . Researchers studying KLHL35 should employ phosphoproteomic approaches to identify potential phosphorylation sites, particularly in regions that might affect protein-protein interactions or subcellular localization. Additionally, since many Kelch-like proteins function as substrate adaptors for E3 ubiquitin ligase complexes, ubiquitination status of KLHL35 itself might serve as a regulatory mechanism affecting its half-life and activity levels within cells.

What are the optimal expression systems for producing recombinant human KLHL35?

For producing recombinant human KLHL35, researchers should consider several expression systems based on their specific experimental needs. Bacterial expression systems such as E. coli can be suitable for structural studies requiring large protein quantities, though care must be taken regarding proper folding of the complex BTB and kelch domains. For studies requiring post-translational modifications and proper folding, mammalian expression systems like HEK293T cells may be more appropriate, despite their relatively lower expression levels compared to bacterial systems . When designing expression constructs, researchers should consider including purification tags that won't interfere with protein function - N-terminal tags are often preferred as the C-terminal β-propeller structure formed by the kelch repeats is crucial for protein-protein interactions. Additionally, expression of individual domains (BTB domain or kelch repeats separately) may be necessary for specific interaction studies, as demonstrated in research with related kelch proteins where MBP-fusion proteins of specific domains were used to characterize functional properties .

How can researchers effectively assess KLHL35 interaction with the cytoskeleton?

Based on findings with related Kelch proteins like KLEIP, researchers investigating KLHL35's potential interactions with cytoskeletal components should employ multiple complementary approaches. Actin co-sedimentation assays represent a fundamental in vitro method, where purified KLHL35 protein is incubated with F-actin, followed by high-speed centrifugation to separate bound from unbound fractions . This technique allows quantitative assessment of binding affinity through detection in supernatant versus pellet fractions. For cellular studies, fluorescently tagged KLHL35 constructs can be expressed in relevant cell lines to visualize co-localization with cytoskeletal elements using confocal microscopy. Advanced techniques like proximity ligation assays would provide higher sensitivity for detecting in situ interactions. Additionally, domain-specific constructs (particularly isolating the kelch-repeat region) should be tested in parallel, as studies with KLEIP demonstrated that the kelch repeats play a critical role in F-actin association while the N-terminal BTB domain participates in dimerization . Researchers should also investigate whether KLHL35 preferentially interacts with specific actin structures or other cytoskeletal components beyond actin.

What antibody validation strategies are essential when studying endogenous KLHL35?

Thorough antibody validation is critical for KLHL35 research to avoid misinterpretation of results due to non-specific binding. Researchers should implement a multi-step validation process similar to that used for other Kelch proteins . First, antibodies should be tested against recombinant KLHL35 and in cells with KLHL35 overexpression to confirm they recognize the target protein at the expected molecular weight. Equally important is validation in KLHL35 knockout or knockdown cells to confirm absence of the specific band. As demonstrated in studies with KLEIP, where antibodies against different regions (N-terminal and C-terminal) helped distinguish between specific and non-specific signals, researchers should consider using multiple antibodies targeting different KLHL35 epitopes . For immunoprecipitation experiments, antibody specificity should be confirmed by mass spectrometry analysis of the immunoprecipitated proteins. Additionally, for immunofluorescence applications, different fixation methods should be tested, as the detection of kelch proteins can be fixation-dependent due to conformational changes, as observed with anti-KLEIP-N antibody which recognized only denatured forms of some proteins .

What cellular processes is KLHL35 likely involved in based on its family members?

Based on functional studies of related Kelch family proteins, KLHL35 may participate in several cellular processes that researchers should investigate. Cell adhesion and cytoskeletal organization are prime candidates, as KLEIP (another Kelch-like protein) localizes to cell-cell adhesion sites and is involved in actin remodeling during cell-cell contact . KLHL35 might also be involved in protein quality control pathways, given that many Kelch-like proteins function as substrate recognition components of E3 ubiquitin ligase complexes that target proteins for degradation, similar to how KLHDC2 has been implicated in targeted protein degradation . Additionally, developmental processes and cell differentiation could be regulated by KLHL35, as observed with PPKL which plays essential roles in parasite differentiation, motility and transmission in Plasmodium . Researchers should employ RNA interference or CRISPR-Cas9 genome editing to generate KLHL35-deficient cell lines, followed by phenotypic analyses focusing on these processes, including assessments of cell morphology, migration, proliferation, and responses to various cellular stresses.

How can researchers identify specific protein binding partners of KLHL35?

To comprehensively identify KLHL35 binding partners, researchers should employ complementary approaches beginning with affinity purification coupled with mass spectrometry (AP-MS). This involves expressing tagged KLHL35 (ensuring the tag doesn't interfere with protein interactions), followed by immunoprecipitation and mass spectrometric analysis of co-purified proteins. BioID or APEX proximity labeling techniques offer advantages for detecting transient or weak interactions by covalently tagging proximal proteins in living cells. For validation of specific interactions, researchers should perform reciprocal co-immunoprecipitation experiments and demonstrate co-localization by microscopy. Domain-specific interaction mapping is crucial, as different domains of Kelch proteins mediate distinct interactions - the BTB domain typically facilitates dimerization while kelch repeats mediate substrate recognition . Additionally, yeast two-hybrid screens can complement these approaches, as demonstrated in the identification of KLEIP through its interaction with ECT2 . When analyzing results, researchers should apply stringent controls and statistical thresholds to distinguish true interactors from contaminants, and consider that interaction partners may vary across cell types and physiological conditions.

What techniques are most effective for studying KLHL35 subcellular localization?

For accurately determining KLHL35 subcellular localization, researchers should implement multiple complementary approaches. Immunofluorescence using validated antibodies against endogenous KLHL35 provides the most physiologically relevant information, but should be performed with multiple fixation protocols as kelch protein detection can be fixation-dependent . For live-cell imaging, expression of fluorescently-tagged KLHL35 (preferably with small tags like mNeonGreen to minimize functional interference) allows dynamic localization studies, though potential overexpression artifacts must be considered. Biochemical fractionation techniques provide quantitative distribution data across cellular compartments and can detect pools of protein not readily visualized by microscopy. When designing these experiments, researchers should examine localization under various cellular conditions that might trigger relocalization, such as cell-cell contact formation, as observed with KLEIP which accumulated at sites of cell-cell adhesion . Super-resolution microscopy techniques like STORM or PALM offer enhanced spatial resolution for precise localization relative to cellular structures. Additionally, domain-specific constructs should be tested to identify which regions determine localization, as different domains may direct the protein to distinct subcellular sites.

What disease associations have been reported for KLHL35 and related Kelch proteins?

While specific disease associations for KLHL35 are not extensively documented in the provided research materials, investigation of potential pathological roles should be guided by the established connections between related Kelch proteins and various disorders. The Kelch protein superfamily has broad implications in human disease , with members functioning in processes like protein degradation that when dysregulated can contribute to pathological conditions. Researchers investigating KLHL35 in disease contexts should conduct transcriptomic and proteomic analyses across tissue samples from various disorders, particularly focusing on conditions involving cytoskeletal abnormalities or cell adhesion defects, given the role of related proteins like KLEIP in these processes . Cancer is a particularly important area for investigation, as some Kelch-like proteins have been implicated in malignant transformation, with altered expression observed in specific cancer types. Additionally, given the conservation of these proteins across species, insights might be gained from model organisms where mutations in kelch-related genes result in developmental abnormalities or functional deficits.

How can researchers effectively use CRISPR-Cas9 gene editing to study KLHL35 function?

CRISPR-Cas9 technology provides powerful approaches for investigating KLHL35 function in cellular and animal models. Researchers should design multiple guide RNAs targeting different exons of KLHL35, preferably early exons to ensure complete functional knockout. For verification of knockout efficiency, both genomic sequencing of the targeted region and Western blot analysis using validated antibodies are essential . Beyond complete gene knockout, researchers should consider more sophisticated CRISPR approaches: knock-in of fluorescent tags at the endogenous locus avoids overexpression artifacts while allowing visualization; conditional knockout systems permit temporal control of gene disruption; and precise point mutations can test the functional importance of specific domains or post-translational modification sites. When analyzing phenotypes of KLHL35-edited cells, researchers should examine processes implicated for related Kelch proteins, including cytoskeletal organization, cell-cell adhesion, and protein degradation pathways . Importantly, rescue experiments with wild-type KLHL35 expression are crucial to confirm that observed phenotypes are specific to KLHL35 loss rather than off-target effects. For organisms where complete knockout may be lethal, heterozygous models or tissue-specific knockout approaches should be considered.

What are the challenges in developing therapeutic approaches targeting KLHL35?

Developing therapeutic strategies targeting KLHL35 presents several research challenges that investigators must address. First, achieving specificity is particularly difficult given the structural similarities between KLHL35 and other Kelch family proteins - researchers must thoroughly characterize binding pockets unique to KLHL35 to avoid off-target effects on related proteins. Small molecule development should focus on compounds that either enhance or inhibit specific protein-protein interactions rather than targeting the entire protein. Recent advances with KLHDC2, where small molecules for targeted protein degradation have been identified , provide a potential template for KLHL35-directed therapeutic development. Second, researchers must establish clear disease contexts where KLHL35 modulation would be beneficial, requiring comprehensive characterization of its expression patterns across healthy and diseased tissues. Third, delivery methods must be optimized for targeting specific tissues where KLHL35 intervention would be therapeutic. For validation of potential therapeutics, researchers should develop cellular and animal models that accurately recapitulate the disease-relevant functions of KLHL35, and establish quantifiable readouts of KLHL35 activity that can serve as pharmacodynamic markers during therapy development.

How do evolutionary considerations inform functional studies of KLHL35?

Evolutionary analysis provides crucial context for KLHL35 research, helping identify conserved functional domains and species-specific adaptations. Researchers should conduct comprehensive phylogenetic comparisons of KLHL35 across species, similar to analyses performed for other Kelch proteins like PPKL, which revealed conservation patterns in kelch repeats with the fourth domain showing highest identity between species . Such analyses can highlight regions under evolutionary pressure, suggesting functional importance. Cross-species conservation studies can also identify potential model organisms for in vivo research, though caution is needed as even closely related proteins can have divergent functions, as observed between KLEIP and Drosophila Kelch despite structural similarities . Additionally, researchers should examine tissue-specific expression patterns across species to identify conserved expression profiles suggesting fundamental functions versus divergent patterns indicating species-specific adaptations. Paralog analysis within the human genome is equally important - comparison with other KLHL family members can reveal unique features of KLHL35. These evolutionary insights should guide experimental design, particularly for structure-function studies and the selection of critical residues for site-directed mutagenesis.

What are the most promising high-throughput screening approaches for identifying KLHL35 modulators?

For identifying modulators of KLHL35 activity, researchers should implement complementary high-throughput screening strategies. Cell-based phenotypic screens represent a powerful approach, where KLHL35 expression or activity is linked to a readily measurable output such as reporter gene expression or cellular phenotype. Researchers could develop cell lines with fluorescent tags on known or predicted KLHL35 substrates to monitor degradation kinetics in response to compound libraries. For protein-protein interaction modulators, split-luciferase assays or FRET-based systems can detect compounds that enhance or disrupt specific KLHL35 interactions. Biochemical screens using purified recombinant KLHL35 can identify direct binders, particularly to specific domains like the kelch repeats or BTB domain. Recent advances in targeted protein degradation approaches with KLHDC2 suggest that PROTAC (Proteolysis Targeting Chimera) technology might be applicable to KLHL35 research, where bifunctional molecules could be screened for their ability to redirect KLHL35 activity toward specific degradation targets. Fragment-based drug discovery approaches may be particularly suitable for the well-defined binding pockets formed by the β-propeller structure of kelch repeats. For all screening approaches, researchers should implement counter-screens against related Kelch proteins to identify KLHL35-selective compounds early in the discovery process.

How can single-cell technologies advance understanding of KLHL35 function in heterogeneous tissues?

Single-cell technologies offer powerful approaches for unraveling KLHL35 function in complex tissue environments where cellular heterogeneity may mask important biological signals. Single-cell RNA sequencing can reveal cell type-specific expression patterns of KLHL35 and correlation with expression of potential interaction partners across developmental stages, disease progression, or in response to various stimuli. This approach is particularly valuable given that some Kelch family proteins show variable expression across cell types, as observed with KLEIP which had highest expression in MDCK cells compared to other cell lines tested . For protein-level analysis, mass cytometry (CyTOF) or imaging mass cytometry using validated KLHL35 antibodies can map protein expression and modification status across tissue architecture while preserving spatial information. Single-cell ATAC-seq can identify cell type-specific regulatory elements controlling KLHL35 expression, while spatial transcriptomics approaches can place KLHL35 expression patterns in the context of tissue microenvironments. For functional studies, CRISPR screens with single-cell readouts can identify genetic interactions with KLHL35 across diverse cell populations. Researchers implementing these technologies should develop computational pipelines capable of integrating multi-modal single-cell data to build comprehensive models of KLHL35 regulation and function within tissue contexts.