Recombinant Candida glabrata Inheritance of peroxisomes protein 2 (INP2)

What is Inheritance of Peroxisomes Protein 2 (INP2) in Candida glabrata?

Inheritance of peroxisomes protein 2 (INP2) is a protein coded by the INP2 gene (also annotated as CAGL0H04455g) in Candida glabrata . It functions as a key component in the machinery that controls peroxisome inheritance during cell division, similar to the related protein Inp1. While Inp1 is involved in peroxisome retention in mother cells through its interaction with Pex3 and plasma membrane components, INP2 appears to play a complementary role in the distribution of these organelles . The protein is part of the complex cellular mechanisms that ensure proper organelle distribution during yeast budding. Understanding INP2 provides insights into fundamental cellular processes of organelle inheritance and may contribute to our knowledge of C. glabrata pathogenicity mechanisms.

How does INP2 compare functionally to the related protein INP1?

The functional comparison between INP2 and INP1 reveals complementary but distinct roles in peroxisome inheritance. Inp1 forms a tethering complex with Pex3 that is essential for peroxisome retention during cell division and for positioning peroxisomes along the mother cell cortex . This tethering occurs via an N-terminal domain of Inp1 that binds PI(4,5)P2 in the plasma membrane and a C-terminal domain that binds Pex3 in the peroxisomal membrane, effectively creating a bridge between these structures . In contrast, while the specific mechanism of INP2 remains less thoroughly characterized in the literature, it appears to function in the opposing process of facilitating peroxisome movement toward the daughter cell. This balanced system of retention (Inp1) and transport (potentially involving INP2) ensures the proper distribution of peroxisomes between mother and daughter cells during yeast budding. Understanding this balance provides crucial insights into organelle inheritance mechanisms that are fundamental to fungal cell biology.

What cellular phenotypes are associated with INP2 dysfunction?

Dysfunction of peroxisome inheritance proteins produces distinct cellular phenotypes that reflect their biological roles. While specific INP2 mutant phenotypes in C. glabrata are not directly described in the provided search results, we can infer likely outcomes based on related proteins. For comparison, deletion of INP1 (inp1Δ) results in a distinctive phenotype where most peroxisomes are present in the bud rather than properly distributed between mother and daughter cells . This demonstrates the critical role of Inp1 in anchoring peroxisomes to the mother cell cortex. By extension, INP2 dysfunction would likely result in the opposite phenotype—retention of peroxisomes in the mother cell with insufficient distribution to daughter cells. Such inheritance defects could potentially affect metabolic functions that rely on peroxisomes, including fatty acid beta-oxidation, detoxification of reactive oxygen species, and other specialized metabolic pathways. These phenotypes provide valuable experimental readouts for researchers studying peroxisome inheritance mechanisms in C. glabrata.

What expression systems are optimal for producing recombinant C. glabrata INP2?

The optimal expression system for recombinant C. glabrata INP2 production is E. coli, as indicated by commercial recombinant INP2 preparations . This prokaryotic expression system offers several advantages for protein production, including rapid growth, high yields, and well-established genetic manipulation protocols. When expressing C. glabrata INP2 in E. coli, researchers should consider codon optimization to account for differences in codon usage bias between the fungal source and bacterial host. The recombinant protein is typically produced with a fusion tag (such as His, GST, or MBP) to facilitate purification and may be stored in a liquid form containing glycerol to maintain stability . For applications requiring post-translational modifications that E. coli cannot provide, researchers might consider yeast-based expression systems like Saccharomyces cerevisiae or Pichia pastoris, which could potentially produce a more native-like protein structure.

What purification strategies yield high-quality INP2 protein preparations?

Purification of recombinant INP2 requires a strategic approach to obtain high-quality protein preparations suitable for downstream applications. For His-tagged INP2, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins provides an effective initial purification step, typically followed by size exclusion chromatography to remove aggregates and improve homogeneity. Based on procedures used for similar proteins, buffer optimization is crucial, with typical buffers containing 20-50 mM phosphate or Tris at pH 7.4-8.0, 150-300 mM NaCl, and potentially small amounts of reducing agents like DTT or β-mercaptoethanol to maintain cysteine residues in reduced form. The addition of 5-10% glycerol in storage buffers helps prevent freeze-thaw damage during storage at -20°C or -80°C . For functional studies, researchers should verify protein activity through binding assays with known interaction partners, such as testing for interaction with Pex3 using methods similar to those employed for Inp1-Pex3 binding studies .

How can researchers assess the functionality of recombinant INP2?

Assessing the functionality of recombinant INP2 requires multi-dimensional approaches that examine both its binding properties and biological activity. In vitro binding assays can evaluate interactions with known or predicted binding partners, particularly components of the peroxisomal machinery such as Pex3. These assays might employ techniques similar to those used for Inp1, including pull-down assays with recombinant Pex3 . Structure-function relationships can be explored by creating truncation mutants or targeted mutations of key motifs, similar to the approach used to identify the LXXLL motif essential for Inp1-Pex3 interaction . Complementation assays in inp2Δ mutant strains provide a powerful method to assess in vivo functionality, where the ability of different recombinant INP2 variants to restore normal peroxisome distribution can be quantified through microscopic analysis of fluorescently labeled peroxisomes. Additionally, localization studies using GFP-tagged INP2 can confirm proper subcellular targeting, while co-immunoprecipitation experiments may identify novel interaction partners in the cellular context.

How does INP2 contribute to interorganellar contact sites in C. glabrata?

The role of INP2 in interorganellar contact sites represents an emerging area of interest in cell biology research. Peroxisomes in yeast interact with multiple cellular structures, including the plasma membrane, ER, vacuole, mitochondria, and lipid bodies, forming specialized membrane contact sites that facilitate communication between organelles . While the components of some interorganellar peroxisomal contact sites have been identified, others remain uncharacterized, including potential contributions of INP2 to these structures. By analogy with Inp1, which forms part of the plasma membrane–peroxisome (PM-PER) contact site through its interaction with both Pex3 on peroxisomes and PI(4,5)P2 on the plasma membrane, INP2 may participate in similar tethering complexes with different specificities or in different cellular contexts . These contact sites likely play roles beyond physical tethering, potentially facilitating lipid transfer, metabolite exchange, or signaling between organelles. Research into INP2's role in these structures could provide insights into the spatial organization of peroxisomal metabolism and its integration with other cellular processes.

What is the relationship between INP2 function and C. glabrata pathogenicity?

The potential relationship between INP2 function and C. glabrata pathogenicity presents an intriguing research direction, though direct evidence is limited in the current literature. C. glabrata has emerged as a major health threat partly due to its ability to acquire resistance to multiple antifungal drug classes . The genomic plasticity of C. glabrata, including enrichment of non-synonymous mutations in genes encoding cell-wall proteins observed in clinical isolates, suggests adaptive processes occurring during infection . While INP2 is not explicitly identified among these genes in the provided search results, disruption of peroxisome homeostasis could potentially affect pathogenicity through several mechanisms. These might include altered metabolism affecting stress responses, changes in cell wall composition or integrity, or modifications in host-pathogen interactions. Research examining INP2 expression or mutations in clinical isolates with different virulence profiles could reveal whether this protein contributes to adaptation during infection. Additionally, studies comparing wild-type and inp2Δ mutant strains in infection models would provide direct evidence regarding the role of INP2 in pathogenicity.

How do INP2 sequence variations among clinical isolates impact protein function?

The impact of INP2 sequence variations among clinical isolates on protein function represents a potentially significant but underexplored area of research. Genome comparisons of C. glabrata serial clinical isolates have revealed an enrichment of non-synonymous changes in genes encoding cell-wall proteins, suggesting selection processes occurring within the human host . While specific INP2 variations are not explicitly described in the provided search results, the observed genetic plasticity in C. glabrata raises the possibility that INP2 might also exhibit functional polymorphisms. Analysis of INP2 sequences across clinical isolates could identify potential hotspots for variation, particularly in domains mediating protein-protein interactions or membrane associations. Functional characterization of these variants through complementation assays, binding studies, and localization experiments would reveal whether they alter peroxisome inheritance patterns or interactions with binding partners. Such research could connect peroxisome inheritance mechanisms to adaptive processes during infection and potentially identify novel intervention targets.

What microscopy techniques are most effective for studying INP2-mediated peroxisome inheritance?

Advanced microscopy techniques offer powerful approaches for investigating INP2-mediated peroxisome inheritance in C. glabrata. Live-cell fluorescence microscopy using peroxisome-targeted fluorescent proteins (such as BFP-PTS1, which targets to peroxisomes via the peroxisomal targeting signal type 1) enables dynamic visualization of peroxisome movements during cell division . This approach can be enhanced by simultaneously labeling the plasma membrane (using markers like GFP-Sso1) and other cellular structures to visualize contacts between peroxisomes and these compartments . For higher-resolution imaging, techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy can resolve subcellular structures beyond the diffraction limit of conventional light microscopy. Time-lapse imaging during budding is particularly valuable for quantifying peroxisome inheritance dynamics, allowing researchers to track the movement of individual peroxisomes and measure retention versus transport rates. Correlation of these microscopic observations with biochemical data on INP2 interactions provides a comprehensive understanding of the molecular mechanisms underlying peroxisome inheritance.

How can researchers generate and validate INP2 knockout strains?

Generating and validating INP2 knockout strains in C. glabrata requires careful molecular genetic approaches. The gene deletion can be achieved through homologous recombination using a deletion cassette containing a selectable marker (typically a drug resistance gene) flanked by sequences homologous to regions upstream and downstream of the INP2 gene. While C. glabrata has lower homologous recombination efficiency than S. cerevisiae, optimized transformation protocols using lithium acetate methods can achieve successful integration. PCR verification with primers binding outside the integration site can confirm correct insertion of the deletion cassette, while reverse transcription PCR or western blotting can verify the absence of INP2 transcript or protein. Phenotypic validation should include microscopic assessment of peroxisome distribution using fluorescently labeled peroxisomes, with the expected phenotype being altered peroxisome inheritance patterns during cell division. Complementation with the wild-type INP2 gene should restore normal peroxisome distribution, confirming that the observed phenotype results specifically from INP2 deletion rather than from secondary mutations or off-target effects.

What bioinformatic approaches can identify functional domains within INP2?

Bioinformatic approaches provide valuable insights into the functional domains of INP2 without requiring extensive experimental work. Sequence alignment tools comparing INP2 across fungal species can identify conserved regions likely to be functionally important, similar to the approach that helped characterize Inp1 domains . Motif analysis may reveal binding sites for interaction partners, such as the LXXLL motif found in Inp1 that mediates binding to Pex3 . Structural prediction algorithms, including AlphaFold2, can generate models of INP2 tertiary structure, potentially highlighting functional surfaces or domains. Hydrophobicity analysis and membrane interaction prediction tools may identify regions that associate with lipid bilayers, similar to the N-terminal PI(4,5)P2-binding domain of Inp1 . Integration of these computational approaches with limited experimental validation (such as testing the effect of mutations in predicted functional regions) can efficiently characterize INP2 domains. This knowledge guides the design of functional studies and the development of tools to manipulate INP2 activity in research contexts.

How does C. glabrata INP2 compare with homologs in other fungal species?

A comparative analysis of C. glabrata INP2 with homologs in other fungal species reveals evolutionary patterns and functional conservation. While specific comparative data for INP2 is not provided in the search results, approaches similar to those used for other peroxisomal proteins can be applied. Sequence alignment and phylogenetic analysis would likely show highest similarity to INP2 proteins in other Candida species and the closely related Saccharomyces genus, with decreasing conservation in more distantly related fungi. Functional domains, particularly those mediating interactions with highly conserved peroxisomal machinery like Pex3, would show greater sequence conservation than regions involved in species-specific functions. Comparison with the well-studied S. cerevisiae INP2 could guide functional investigations in C. glabrata, while identifying divergent regions might highlight adaptations specific to C. glabrata's lifestyle as an opportunistic pathogen. Such comparative analyses not only illuminate evolutionary relationships but also help identify critical functional regions that could serve as targets for experimental manipulation or potential therapeutic intervention.

What animal models are suitable for studying C. glabrata peroxisome dynamics?

Selecting appropriate animal models for studying C. glabrata peroxisome dynamics requires balancing pathophysiological relevance with experimental accessibility. Murine models of C. glabrata infection represent the most widely used system, with immunocompromised mice providing a suitable environment for fungal colonization and persistence . These models can be adapted to study peroxisome dynamics by using fluorescently tagged peroxisomal markers in the infecting C. glabrata strains, though in vivo imaging may present technical challenges. For more controlled studies of peroxisome inheritance, ex vivo systems using infected macrophages or epithelial cells allow higher-resolution microscopy while maintaining host-relevant conditions. The choice between systemic infection models (typically via intravenous injection) versus mucosal colonization models (oral or vaginal) should be guided by the specific research questions, as peroxisome dynamics might differ between these infection contexts. Regardless of the model selected, comparison of wild-type and inp2Δ strains would reveal the impact of peroxisome inheritance defects on colonization, persistence, and virulence in host environments.

How does the mutator phenotype in C. glabrata affect INP2 genetic stability?

The prevalent mutator phenotype identified in C. glabrata clinical isolates raises important questions about INP2 genetic stability. Approximately 55% of clinical isolates carry nonsynonymous mutations in MSH2, a key component of the mismatch repair system, resulting in elevated mutation rates and facilitating rapid adaptation to selective pressures such as antifungal treatment . This hypermutator state could potentially affect the INP2 gene, leading to functional variants with altered peroxisome inheritance properties. The enrichment of nonsynonymous mutations in cell wall proteins observed in clinical isolates suggests that genes encoding surface-exposed or host-interface proteins experience selective pressure during infection . Whether INP2 falls into this category remains an open question, but researchers working with clinical isolates should consider sequencing INP2 to identify potential variations. Studies comparing INP2 sequence and function between MSH2-defective strains and mismatch repair-proficient strains across multiple passages or during antifungal selection could reveal whether peroxisome inheritance mechanisms represent an adaptive target during infection or treatment.

What are the promising research gaps in understanding INP2 function?

Several promising research gaps exist in our understanding of INP2 function that merit further investigation. First, the specific molecular mechanisms by which INP2 contributes to peroxisome inheritance remain incompletely characterized, particularly in comparison to the better-understood Inp1-Pex3 tethering complex . Elucidating these mechanisms would advance fundamental knowledge of organelle inheritance. Second, the regulation of INP2 expression and activity under different growth conditions or stressors presents an important area for exploration, potentially revealing how peroxisome dynamics adapt to changing environments. Third, the possible role of INP2 in interorganellar communication beyond peroxisome inheritance remains largely unexplored, despite growing recognition of peroxisomes as hubs in cellular metabolic networks. Fourth, potential connections between INP2 function and C. glabrata pathogenicity or stress responses represent an intriguing direction for investigation, especially given the organism's importance as an opportunistic pathogen . Finally, the evolution of peroxisome inheritance mechanisms across fungal species offers opportunities for comparative studies that could reveal both conserved principles and species-specific adaptations.

How might INP2 function be leveraged for antifungal development?

The potential to leverage INP2 function for antifungal development presents an innovative approach to addressing the growing challenge of C. glabrata infections. C. glabrata has emerged as a major health threat partly due to its ability to acquire resistance to multiple drug classes, including triazoles and echinocandins . Novel targets unrelated to current antifungals are therefore valuable for developing new therapeutic approaches. While peroxisomes are present in both fungal and human cells, the specific machinery of peroxisome inheritance differs significantly, potentially providing fungal-specific targets. Compounds that selectively interfere with INP2 function or its interactions with binding partners could disrupt peroxisome inheritance, potentially compromising fungal metabolism, stress responses, or cell division. A target-based drug discovery approach would begin with high-throughput screens for compounds that disrupt INP2 interactions, followed by medicinal chemistry optimization and validation in cellular and animal models. Alternatively, exploring whether existing drugs might have secondary effects on peroxisome dynamics could identify repurposing opportunities. The development of such novel antifungals would be particularly valuable given the concerning prevalence of mutator phenotypes in C. glabrata that facilitate rapid acquisition of resistance to current drugs .

What technological advances would most benefit INP2 research?

Several technological advances would significantly benefit INP2 research and accelerate progress in understanding this protein's functions. Improved CRISPR-Cas9 systems optimized for C. glabrata would facilitate more efficient gene editing, enabling rapid generation of mutants and tagged proteins for functional studies. Advanced super-resolution microscopy techniques with increased temporal resolution would allow real-time visualization of peroxisome dynamics during cell division with unprecedented detail. Proximity labeling methods such as BioID or APEX, adapted for fungal systems, could identify the INP2 interactome in different cellular contexts, revealing previously unknown binding partners and functional connections. Microfluidic systems coupled with live-cell imaging would enable long-term observation of peroxisome inheritance across multiple generations under controlled conditions that can mimic different host environments. Finally, improved recombinant protein expression systems specifically optimized for difficult-to-express fungal proteins would facilitate structural studies and biochemical characterization of INP2. These technological advances, applied in combination, would provide comprehensive insights into INP2 function and its role in C. glabrata biology and pathogenicity.