Recombinant Neosartorya fumigata Eukaryotic translation initiation factor 3 subunit G (tif35)

What is Neosartorya fumigata and how is it related to Aspergillus fumigatus?

Neosartorya fumigata is the sexual teleomorph (reproductive form) of Aspergillus fumigatus, a ubiquitous environmental mold. Historically, A. fumigatus was considered asexual until studies revealed a heterothallic breeding system. As noted in research, "Neosartorya sp. are the complementary mating types of Aspergillus and are required for sex to occur" . This relationship is crucial for understanding the complete lifecycle and genetic diversity of this organism. The teleomorph-anamorph relationship contributes to the pathogen's adaptability and virulence potential, with Neosartorya species emerging as causes of invasive infections in humans, including severe conditions like acute respiratory distress syndrome (ARDS) .

What is the role of eukaryotic translation initiation factor 3 subunit G (tif35) in fungal protein synthesis?

Eukaryotic translation initiation factor 3 subunit G (tif35) is a component of the eIF3 complex, which plays a crucial role in translation initiation. As part of the eIF3 complex, tif35 contributes to scaffolding and regulatory functions similar to other eIF3 subunits like eIF3b, which has been described as "the main scaffolding subunit in the eIF3 complex" . The eIF3 complex facilitates the recruitment of mRNA to the ribosome and assists in the assembly of the translation initiation complex. In fungi, tif35 participates in regulating which mRNAs are translated, thereby influencing cellular protein composition and function. This regulation is particularly important during stress responses and developmental transitions, which are critical for fungal pathogenicity and survival under changing environmental conditions.

How is tif35 gene expression regulated in Neosartorya fumigata?

Tif35 expression in Neosartorya fumigata appears to be regulated in response to environmental conditions, as evidenced by studies showing TIF35 downregulation under certain experimental conditions . The regulation likely involves multiple mechanisms:

• Transcriptional regulation through specific transcription factors that respond to stress conditions

• Post-transcriptional regulation via RNA-binding proteins or non-coding RNAs

• Epigenetic modifications affecting chromatin accessibility

• Signal transduction pathways that connect environmental cues to gene expression changes

The dynamic regulation of tif35 expression may be particularly important during infection processes, as the pathogen must adapt to changing host environments and stress conditions. This versatility in gene expression contributes to the fungus's ability to survive in diverse ecological niches and within human hosts.

What are the optimal expression systems for producing recombinant Neosartorya fumigata tif35?

For optimal expression of recombinant Neosartorya fumigata tif35, researchers should consider several expression systems, each with distinct advantages:

When expressing tif35 in E. coli systems, consider these optimization strategies:

• Expression at lower temperatures (16-20°C) to enhance protein solubility

• Use of solubility-enhancing fusion tags (His6, GST, SUMO)

• Codon optimization for the expression host

• Co-expression with chaperones to improve folding

The specific choice should be guided by the intended experimental application, with bacterial systems being sufficient for structural studies and more complex eukaryotic systems necessary when authentic post-translational modifications are required.

What purification strategies yield functional tif35 protein?

To obtain functional tif35 protein while preserving its native conformation, a multi-step purification strategy is recommended:

• Initial Capture:

  • For His-tagged constructs: Immobilized Metal Affinity Chromatography (IMAC) with Ni-NTA resin

  • For GST-tagged proteins: Glutathione-agarose affinity chromatography

• Intermediate Purification:

  • Ion exchange chromatography (typically Q or SP sepharose)

  • Heparin affinity chromatography (leveraging tif35's RNA-binding properties)

• Polish Purification:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Remove fusion tags using specific proteases (TEV, PreScission)

• Buffer Optimization:

  • pH range: 7.0-8.0 (phosphate or Tris-based buffers)

  • Salt concentration: 150-300 mM NaCl

  • Stabilizing agents: 5-10% glycerol, 1-5 mM DTT

  • Consider adding 0.1 mM EDTA to prevent metal-catalyzed oxidation

A critical quality control step is to assess the functional activity of the purified protein through RNA binding assays and protein interaction studies to confirm that the purification process has preserved the biological activity of tif35.

What functional assays can verify the activity of recombinant tif35?

To verify the functional activity of recombinant Neosartorya fumigata tif35, several complementary assays should be employed:

• RNA Binding Assays:

  • Electrophoretic Mobility Shift Assay (EMSA) to detect tif35-RNA interactions

  • Filter binding assays with radiolabeled RNA

  • Fluorescence anisotropy to measure binding affinities

• Protein-Protein Interaction Studies:

  • Pull-down assays with other eIF3 subunits, particularly eIF3b

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Translation Assays:

  • In vitro translation systems supplemented with recombinant tif35

  • Reconstitution of 43S pre-initiation complex formation

  • Assessment of tif35's contribution to translation of reporter mRNAs

• Structural Integrity Tests:

  • Circular dichroism spectroscopy to verify secondary structure

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to confirm proper domain folding

Each assay provides different insights into tif35 functionality, and combining multiple approaches provides the most comprehensive validation of the recombinant protein's activity.

How does tif35 contribute to the eIF3 complex structure and function?

Tif35 plays several critical roles in the eIF3 complex structure and function in Neosartorya fumigata:

• Structural Organization:

  • Tif35 likely interacts directly with eIF3b, which serves as "the main scaffolding subunit in the eIF3 complex"

  • Forms part of the RNA-binding surface of the eIF3 complex

  • Contributes to maintaining the three-dimensional architecture necessary for function

• RNA Recognition:

  • Contains an RNA Recognition Motif (RRM) that binds specific mRNA features

  • Assists in positioning mRNA on the 40S ribosomal subunit

  • May contribute to selective mRNA recruitment during translation initiation

• Regulatory Functions:

  • Participates in translational control during stress conditions

  • Mediates responses to environmental changes through altered translation patterns

  • May facilitate selective translation of virulence-related mRNAs during infection

• Complex Assembly:

  • Serves as an assembly platform for specific eIF3 subunits

  • Contributes to the stability of the multi-subunit complex

  • Coordinates interactions with other initiation factors

Understanding these roles is crucial for developing targeted approaches to study tif35 function in pathogenic fungi and potentially for designing selective inhibitors as antifungal agents.

What signaling pathways interact with tif35 during fungal stress response?

Tif35 intersects with several signaling pathways during fungal stress response, which reflects its role in translation regulation under changing environmental conditions:

• Target of Rapamycin (TOR) Pathway:

  • Coordinates protein synthesis with nutrient availability

  • TOR kinase activity affects translation initiation factor function

  • Links cellular energy status to translation rates through eIF3 regulation

• Mitogen-Activated Protein Kinase (MAPK) Cascades:

  • Transmit stress signals to the translation machinery

  • May phosphorylate tif35 or other eIF3 subunits to modulate activity

  • Particularly important during cell wall stress and osmotic challenges

• Integrated Stress Response (ISR):

  • Regulates translation during various cellular stresses

  • Affects eIF3 function through interactions with eIF2

  • Modulates the translation of specific stress-responsive mRNAs

• Hypoxia Response Pathway:

  • Adapts translation to low-oxygen environments encountered during infection

  • Alters the spectrum of translated mRNAs to favor survival proteins

  • TIF35 has been observed to be downregulated under certain stress conditions

• Heat Shock Response:

  • Coordinates translation of heat shock proteins and chaperones

  • Involves specific interactions between heat shock factors and the translation machinery

  • Critical for protein homeostasis during temperature fluctuations

These pathways represent potential targets for intervention in fungal infections, as disrupting the ability of Neosartorya fumigata to adapt to host-imposed stresses could reduce its pathogenicity.

How does tif35 influence selective mRNA translation during infection?

During infection, tif35 likely plays a crucial role in selective mRNA translation that enables Neosartorya fumigata to adapt to the host environment:

• Virulence Factor Expression:

  • Prioritizes translation of mRNAs encoding invasive enzymes and toxins

  • Helps coordinate the expression of virulence factors with environmental cues

  • May recognize specific features in virulence factor mRNAs

• Stress Response Regulation:

  • Facilitates the translation of stress-protective factors when encountering host defenses

  • Diverts translation machinery from housekeeping functions to survival proteins

  • This adaptation is critical during pulmonary infections, where Neosartorya species can cause severe conditions like ARDS

• Metabolic Adaptation:

  • Adjusts the proteome to utilize available nutrients in the host environment

  • Shifts between fermentation and respiration based on oxygen availability

  • Similar adaptations have been observed in yeast models where TIF35 expression changes correlated with metabolic adjustments

• Immune Evasion:

  • Enables rapid translation reprogramming in response to host immune detection

  • Facilitates production of proteins that interfere with host defense mechanisms

  • Contributes to the pathogen's ability to persist despite immune pressure

• Biofilm Formation:

  • Supports translation of mRNAs required for adhesion and extracellular matrix production

  • Coordinates protein synthesis during the transition to biofilm lifestyle

  • Enhances resistance to antifungal drugs and host defenses

Understanding these selective translation mechanisms could provide insights into Neosartorya fumigata pathogenicity and potential intervention strategies.

How conserved is tif35 structure and function across fungal pathogens?

The conservation of tif35 across fungal pathogens reflects its essential role in translation initiation:

The functional conservation appears strong despite sequence divergence, as tif35 must maintain its core role in translation initiation across species. This conservation pattern presents both challenges and opportunities for developing specific antifungal approaches targeting tif35.

What structural differences exist between fungal tif35 and human eIF3g?

Several structural differences between fungal tif35 and human eIF3g have significant implications for research and therapeutic development:

• RNA Recognition Motif (RRM):

  • While the core RRM fold is conserved, specific residues that contact RNA differ

  • Fungal tif35 RRMs typically have more basic residues in RNA-binding loops

  • These differences affect RNA binding specificity and affinity

• Protein Interaction Surfaces:

  • Interface residues for eIF3b binding show significant variation

  • Human eIF3g contains unique motifs for interaction with mammalian-specific partners

  • Fungal tif35 has evolved specific interfaces for fungal translation factors

• Regulatory Regions:

  • N-terminal extensions differ substantially between human and fungal proteins

  • Different phosphorylation sites reflect divergent regulatory mechanisms

  • Fungal-specific regulatory elements that respond to environmental stresses

• Disordered Regions:

  • Different patterns of intrinsically disordered regions

  • These regions often mediate species-specific protein-protein interactions

  • They provide conformational flexibility important for complex assembly

• Surface Charge Distribution:

  • Distinct electrostatic surface potentials affect molecular interactions

  • Fungal tif35 typically has more positively charged patches

  • These charge differences create potential binding pockets for selective targeting

These structural differences could be exploited for the development of selective antifungal compounds that target fungal tif35 without affecting human eIF3g.

How does tif35 contribute to Neosartorya fumigata virulence and pathogenicity?

Tif35 likely contributes to Neosartorya fumigata virulence and pathogenicity through multiple mechanisms:

• Adaptation to Host Environment:

  • Regulates translation to adapt to the nutrient-limited and hostile host environment

  • Facilitates protein synthesis under the stress conditions encountered during infection

  • This adaptation is particularly important in pulmonary infections, where Neosartorya species can cause severe conditions like ARDS

• Virulence Factor Expression:

  • Controls the translation of mRNAs encoding hydrolytic enzymes, toxins, and adhesins

  • Coordinates expression of virulence factors with environmental cues

  • Ensures appropriate timing of virulence factor production during infection progression

• Stress Resistance:

  • Mediates translation reprogramming in response to host immune attacks

  • Facilitates synthesis of proteins that detoxify reactive oxygen species

  • Contributes to thermal stress adaptation, important during fever response

• Morphological Transitions:

  • Supports protein synthesis required for hyphal growth and tissue invasion

  • Regulates translation during the transition between growth forms

  • These morphological changes are critical for tissue penetration and dissemination

• Biofilm Formation:

  • Facilitates translation of proteins involved in adhesion and extracellular matrix production

  • Contributes to the development of drug-resistant biofilm communities

  • Enhances survival within the host through community-based protection

These contributions make tif35 an important factor in the pathogen's ability to cause invasive fungal infections, particularly in immunocompromised individuals.

Could tif35 serve as a target for novel antifungal therapies?

Tif35 presents several characteristics that make it a potentially valuable target for novel antifungal therapies:

While significant research and development would be required, tif35 represents a promising target for next-generation antifungal therapeutics, particularly for invasive Neosartorya infections that can cause severe conditions like ARDS .

How can CRISPR-Cas9 technology be applied to study tif35 function in Neosartorya fumigata?

CRISPR-Cas9 technology offers powerful approaches for investigating tif35 function in Neosartorya fumigata, with several specialized strategies:

• Conditional Depletion Systems:

  • Since complete deletion of tif35 would likely be lethal, conditional approaches are essential

  • Inducible promoter replacement to control tif35 expression levels

  • Auxin-inducible degron (AID) tagging for rapid protein depletion

  • Temperature-sensitive degrons for temporal control

• Domain-Specific Modifications:

  • Precise editing of RNA-binding domains to alter specificity

  • Mutation of interaction surfaces with other eIF3 subunits

  • Introduction of specific phosphomimetic mutations to study regulation

  • Creation of chimeric proteins with domains from other species

• Genomic Tagging Strategies:

  • C-terminal fluorescent protein fusions for localization studies

  • Split fluorescent protein complementation to visualize interactions

  • Affinity tags for purification of native complexes

  • Proximity-labeling tags to identify the tif35 interactome

• Specialized Delivery Methods:

  • Ribonucleoprotein (RNP) delivery to reduce off-target effects

  • Optimized transformation protocols for Neosartorya fumigata

  • Transient expression systems for Cas9 to minimize genomic integration

  • Base editing approaches for precise nucleotide changes

• Experimental Design Considerations:

  • Use of multiple guide RNAs to minimize off-target effects

  • Careful selection of PAM sites to ensure efficient editing

  • Inclusion of reconstitution controls to confirm phenotype specificity

  • Integration of complementary techniques (RNA-seq, proteomics) to characterize effects

These CRISPR-based approaches provide unprecedented precision for manipulating tif35 in its native context, enabling detailed functional studies of this important translation factor.

What advanced imaging techniques can visualize tif35 dynamics during translation?

Advanced imaging techniques offer powerful approaches to visualize tif35 dynamics during translation in Neosartorya fumigata:

• Single-Molecule Fluorescence Techniques:

  • Single-molecule FRET (smFRET) to monitor conformational changes

  • Total internal reflection fluorescence (TIRF) microscopy for surface-immobilized complexes

  • These approaches can reveal the kinetics of tif35 association with ribosomes and other factors

• Super-Resolution Microscopy:

  • Stimulated emission depletion (STED) microscopy for subcellular localization

  • Photoactivated localization microscopy (PALM) for nanoscale resolution

  • Stochastic optical reconstruction microscopy (STORM) to visualize tif35 clusters

  • These techniques overcome the diffraction limit to provide detailed spatial information

• Live-Cell Imaging Approaches:

  • Fluorescence recovery after photobleaching (FRAP) to measure mobility

  • Fluorescence correlation spectroscopy (FCS) for diffusion and binding kinetics

  • These methods provide insights into tif35 dynamics in living fungal cells

• Multi-Color Imaging:

  • Simultaneous visualization of tif35 with other translation factors

  • Colocalization analysis with ribosomes and mRNA

  • Investigation of spatiotemporal coordination during translation initiation

• Correlative Light and Electron Microscopy (CLEM):

  • Combines fluorescence imaging with electron microscopy

  • Provides ultrastructural context for tif35 localization

  • Reveals the association of tif35 with specific cellular compartments

These imaging approaches, combined with appropriate fluorescent tagging strategies, can provide unprecedented insights into the dynamics of tif35 during translation initiation in Neosartorya fumigata.

What omics approaches best characterize the impact of tif35 perturbation?

Multi-omics approaches provide comprehensive insights into the consequences of tif35 perturbation in Neosartorya fumigata:

• Translatomics:

  • Ribosome profiling to map ribosome positions on mRNAs genome-wide

  • Polysome profiling to identify mRNAs with altered translation efficiency

  • These techniques directly assess the primary function of tif35 in translation

• Proteomics:

  • Quantitative proteomics using iTRAQ or TMT labeling to measure protein abundance changes

  • Phosphoproteomics to identify signaling pathways affected by tif35 perturbation

  • Protein-protein interaction studies using affinity purification-mass spectrometry

  • These approaches capture the consequences of altered translation on the proteome

• Transcriptomics:

  • RNA-seq to identify compensatory transcriptional responses

  • CLIP-seq to map direct RNA interactions of tif35

  • Long-read sequencing to detect splicing changes

  • These methods reveal both direct tif35-RNA interactions and indirect effects

• Metabolomics:

  • Targeted and untargeted metabolomics to detect metabolic pathway alterations

  • Stable isotope labeling to measure flux changes

  • These approaches capture the downstream consequences of proteome changes

• Integrated Multi-omics Analysis:

  • Correlation analysis across different data types

  • Network-based integration to identify affected pathways

  • Machine learning approaches to predict functional relationships

  • Similar integrated approaches have been used to study "protein-metabolite joint pathway analysis" in other contexts

The integration of these omics techniques provides a systems-level understanding of tif35 function and how its perturbation affects cellular processes in Neosartorya fumigata.