Recombinant Dictyostelium discoideum Putative uncharacterized protein DDB_G0286829 (DDB_G0286829)

How is DDB_G0286829 expressed and purified for research applications?

For research applications, DDB_G0286829 can be produced as a recombinant protein expressed in E. coli with an N-terminal His-tag. The expression system yields a full-length protein (amino acids 1-193) that can be purified using standard affinity chromatography techniques targeting the His-tag .

The purified protein is typically prepared as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE analysis. For reconstitution, researchers should:

• Briefly centrifuge the vial before opening to collect the product at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50%) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Store at -20°C or -80°C for maximum stability

The protein is supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during storage and reconstitution .

What is currently known about the functional role of DDB_G0286829?

• The protein has been identified in phosphoproteomic analysis studies, suggesting it undergoes phosphorylation and may participate in signaling pathways

• It appears in research related to cyclic GMP-mediated autophagy induction, indicating a potential role in autophagy processes

• As a Dictyostelium protein, it exists in an organism known for unique protein aggregation resistance properties, particularly for proteins with extended polyglutamine tracts

While direct experimental evidence for its function is limited, these contextual associations provide starting points for hypothesis generation and experimental design. Researchers should approach functional characterization through multiple methodologies, including sequence homology analysis, protein interaction studies, and phenotypic analysis of knockout or overexpression models.

What experimental approaches would be appropriate for initial characterization of DDB_G0286829?

For initial characterization of an uncharacterized protein like DDB_G0286829, researchers should consider a multi-faceted approach:

• Bioinformatic analysis:

  • Sequence homology searches against characterized proteins

  • Domain prediction and structural modeling

  • Phylogenetic analysis across species

  • Prediction of post-translational modification sites

• Expression pattern analysis:

  • Temporal expression during Dictyostelium development

  • Spatial expression in different cellular compartments

  • Regulation under different stress conditions (particularly mechanical stress and osmotic shock, given the research context)

• Protein interaction studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation with potential partners

  • Proximity labeling approaches (BioID, APEX)

  • Mass spectrometry-based interactome analysis

• Loss-of-function studies:

  • CRISPR-Cas9 knockout generation

  • RNAi-based knockdown

  • Phenotypic characterization of mutants

• Gain-of-function studies:

  • Overexpression analysis

  • Tagged protein expression for localization studies

  • Structure-function analysis through mutagenesis

These approaches provide complementary data to develop a comprehensive understanding of the protein's biological role.

What evidence exists for DDB_G0286829's potential involvement in autophagy pathways?

DDB_G0286829 appears in research literature concerning cyclic GMP-mediated autophagy induction in Dictyostelium discoideum. While direct evidence for its role in autophagy is limited, the protein was identified in phosphoproteomic analysis in a study examining signaling pathways linked to autophagy induction .

The relationship between mechanical stimulation, cyclic GMP signaling, and autophagy provides context for DDB_G0286829's potential involvement. This research demonstrated that:

• Sustained compression and hyper-osmotic stress can stimulate production of cyclic GMP (cGMP)

• The cGMP analogue 8Br-cGMP potently induced autophagy in a dose-responsive manner

• Phosphoproteomic analysis identified candidate proteins that could facilitate cGMP-induced autophagy

While the specific role of DDB_G0286829 was not explicitly defined, its appearance in this context suggests it may be phosphorylated in response to cGMP signaling or mechanical stimulation, potentially contributing to autophagy regulation.

To investigate this connection further, researchers could:

• Examine DDB_G0286829 phosphorylation status in response to autophagy inducers

• Determine if DDB_G0286829 knockout affects autophagy markers under various stimuli

• Test for interactions between DDB_G0286829 and known autophagy proteins

How might DDB_G0286829 relate to Dictyostelium's unique protein aggregation resistance properties?

Dictyostelium discoideum possesses remarkable resistance to protein aggregation, particularly for proteins with long polyglutamine tracts that typically aggregate in other organisms . This property makes it an interesting model for studying neurodegenerative diseases characterized by protein aggregation.

While the search results don't directly link DDB_G0286829 to this aggregation resistance, several hypotheses can be proposed:

• DDB_G0286829 might function within Dictyostelium's protein quality control machinery that prevents aggregation

• The protein could be involved in cellular stress responses that help maintain proteostasis

• DDB_G0286829 might interact with proteins containing polyglutamine tracts to prevent their aggregation

Research has shown that when polyglutamine-expanded Huntingtin exon 1 (GFPHtt Q103) was expressed in Dictyostelium, it remained soluble, unlike in yeast and human cells where it readily aggregated . This suggests Dictyostelium has evolved specialized mechanisms to maintain protein solubility.

To investigate DDB_G0286829's potential role in this phenomenon, researchers could:

• Test whether DDB_G0286829 knockout affects the solubility of aggregation-prone proteins

• Examine if DDB_G0286829 physically interacts with polyglutamine-containing proteins

• Investigate whether DDB_G0286829 expression is altered during proteotoxic stress

What can phosphoproteomic data reveal about DDB_G0286829's potential function?

Phosphoproteomic analysis provides valuable insights into protein function by identifying phosphorylation sites and their dynamics under different conditions. For DDB_G0286829, phosphoproteomic data from studies on cyclic GMP-mediated autophagy induction offers potential functional clues .

The phosphoproteomic analysis methodologies described in the research included:

• Protein sample preparation and solubilization

• Sample reduction and alkylation

• Trypsin digestion

• Desalting and protein fragment purification

• IMAC purification for phosphopeptide enrichment

• LC-MS/MS analysis

• Mass-spectrometry analysis

While specific phosphorylation sites on DDB_G0286829 were not detailed in the available search results, its appearance in this dataset suggests it undergoes phosphorylation that may be regulated by cGMP signaling or mechanical stimulation.

To leverage phosphoproteomic data for functional insights:

• Identify specific phosphorylation sites on DDB_G0286829

• Determine which kinases might target these sites

• Examine how phosphorylation status changes under different conditions

• Create phosphomimetic and phosphorylation-deficient mutants to test functional significance

• Integrate phosphorylation data with protein interaction networks

This approach could reveal how DDB_G0286829 is regulated and its potential role in signaling pathways.

What structural predictions can be made based on DDB_G0286829's amino acid sequence?

Based on the amino acid sequence of DDB_G0286829, several structural predictions and observations can be made:

• Amino acid composition analysis: The sequence contains a high proportion of asparagine (N) and serine (S) residues, particularly in the N-terminal and middle regions . This suggests potential regions of low complexity that might be intrinsically disordered.

• Potential membrane association: The C-terminal region of the protein contains several hydrophobic residues (e.g., "FLVGASASFGISIGMFYF"), which might indicate a membrane-associated domain or transmembrane region .

• Post-translational modification sites: The abundance of serine residues suggests numerous potential phosphorylation sites, consistent with the protein's identification in phosphoproteomic studies .

• Repetitive elements: The sequence contains repetitive stretches, particularly of asparagine (N) residues, which might form specialized structural elements or interaction surfaces .

Without experimental structural data, researchers should use structural prediction tools to generate hypotheses about:

• Secondary structure elements

• Disorder prediction

• Domain organization

• Potential binding interfaces

• Transmembrane regions

These predictions can guide experimental approaches to determine the actual structure and inform structure-function relationship studies.

What challenges exist in characterizing putative uncharacterized proteins like DDB_G0286829?

Characterizing putative uncharacterized proteins presents several challenges that researchers should consider when studying DDB_G0286829:

• Limited reference information: Without characterized homologs or established functions, researchers lack reference points for experimental design and interpretation.

• Potential for novel functions: The protein may possess functions not previously described, requiring innovative approaches to discover and validate.

• Expression and solubility issues: Recombinant expression may not replicate native folding or post-translational modifications, potentially masking function.

• Context-dependent activity: The protein may function only in specific cellular contexts, developmental stages, or stress conditions.

• Functional redundancy: Other proteins might compensate for its loss in knockout studies, obscuring phenotypes.

• Technical challenges:

  • Generating specific antibodies for an uncharacterized protein

  • Determining appropriate assays without functional knowledge

  • Optimizing conditions for activity assays

  • Interpreting high-throughput data without established biological context

• Integration with existing knowledge: Placing new findings about the protein into the broader understanding of Dictyostelium biology

Addressing these challenges requires a combination of hypothesis-driven approaches and unbiased screening methods to gradually build a functional profile of the protein.

What protocols are recommended for reconstitution and handling of recombinant DDB_G0286829?

For optimal handling of recombinant DDB_G0286829, researchers should follow these protocols:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to bring the lyophilized powder to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Allow the protein to dissolve completely at room temperature with gentle agitation

• Add glycerol to a final concentration of 5-50% (recommended: 50%)

• Aliquot into small volumes to minimize freeze-thaw cycles

Storage Recommendations:

• Store reconstituted protein at -20°C/-80°C for long-term storage

• For working stocks, store aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they may lead to protein denaturation and loss of activity

Handling Guidelines:

• Use appropriate buffer conditions (Tris/PBS-based, pH 8.0) for dilutions

• Include protease inhibitors when working with cell lysates to prevent degradation

• When performing assays, consider the presence of the His tag, which may affect certain interactions

• For maximum stability, handle the protein on ice when thawed

Following these protocols will help maintain protein integrity and maximize experimental reproducibility when working with recombinant DDB_G0286829.

What analytical techniques are most appropriate for studying the properties and interactions of DDB_G0286829?

To comprehensively study DDB_G0286829, researchers should consider multiple analytical techniques:

Structural and Biophysical Techniques:

• Circular Dichroism (CD): To assess secondary structure elements and thermal stability

• Size Exclusion Chromatography (SEC): To determine oligomeric state and hydrodynamic properties

• Differential Scanning Fluorimetry (DSF): To identify stabilizing buffer conditions and potential ligands

• Nuclear Magnetic Resonance (NMR): Particularly useful if regions of the protein are intrinsically disordered

• Small Angle X-ray Scattering (SAXS): For low-resolution structural information in solution

Interaction Studies:

• Pull-down Assays: Using the His-tag for affinity purification of interaction partners

• Surface Plasmon Resonance (SPR): For quantitative binding analysis

• Bio-Layer Interferometry (BLI): Alternative to SPR for kinetic measurements

• Isothermal Titration Calorimetry (ITC): For thermodynamic characterization of binding events

• Crosslinking Mass Spectrometry (XL-MS): To capture transient interactions

Functional Assays:

• Phosphorylation Assays: Given its identification in phosphoproteomic studies

• Autophagy Assays: To investigate potential roles in autophagy pathways

• Stress Response Assays: Particularly focusing on mechanical stress and osmotic shock

• Protein Aggregation Assays: To test potential roles in preventing protein aggregation

Cellular Localization:

• Immunofluorescence: Using antibodies against the protein or its tag

• Subcellular Fractionation: Combined with Western blotting

• Live Cell Imaging: With fluorescently tagged protein

Each technique provides complementary information that, when integrated, can reveal the protein's properties and functions.

How can phosphorylation analysis be optimized for studying DDB_G0286829?

Given DDB_G0286829's appearance in phosphoproteomic studies, optimizing phosphorylation analysis is crucial for understanding its regulation and function:

Sample Preparation Optimization:

• Rapid sample processing: Minimize time between cell harvesting and protein extraction to preserve phosphorylation states

• Phosphatase inhibitors: Include cocktails containing sodium fluoride, sodium orthovanadate, and β-glycerophosphate

• Lysis buffer optimization: Use buffers that efficiently solubilize membrane-associated proteins if DDB_G0286829 has membrane interactions

• Stimulus timing: Capture both rapid (seconds to minutes) and sustained (hours) phosphorylation events

Enrichment Strategies:

• IMAC (Immobilized Metal Affinity Chromatography): Using Fe3+ or Ga3+ for phosphopeptide enrichment

• Titanium dioxide (TiO2) chromatography: Alternative enrichment method with complementary specificity

• Phospho-specific antibodies: For targeted analysis of specific sites once identified

• Sequential elution from IMAC (SIMAC): To separate mono- and multi-phosphorylated peptides

Mass Spectrometry Approaches:

• Data-dependent acquisition (DDA): For discovery of phosphorylation sites

• Selected/Multiple reaction monitoring (SRM/MRM): For targeted quantification of specific phosphosites

• Parallel reaction monitoring (PRM): For improved selectivity in targeted analysis

• Data-independent acquisition (DIA): For comprehensive phosphoproteome coverage

Data Analysis Considerations:

• Site localization algorithms: Such as Ascore or ptmRS to accurately assign phosphorylation sites

• Quantification methods: Label-free, SILAC, or TMT approaches depending on experimental design

• Motif analysis: To identify potential kinases responsible for phosphorylation

• Integration with protein-protein interaction data: To place phosphorylation events in signaling networks

The phosphoproteomic workflow described in the search results includes sample solubilization, reduction and alkylation, trypsin digestion, desalting, IMAC purification, and LC-MS/MS analysis , which provides a foundation for DDB_G0286829 phosphorylation studies.

What approaches can be used to investigate DDB_G0286829's potential role in autophagy?

To investigate DDB_G0286829's potential role in autophagy, researchers should employ multiple complementary approaches:

Genetic Manipulation Approaches:

• Gene knockout/knockdown: Generate DDB_G0286829-deficient Dictyostelium cells to assess effects on:

  • Basal autophagy levels

  • Autophagy induction under starvation

  • Response to mechanical stimulation and cGMP treatment

  • Response to hyper-osmotic stress

• Overexpression studies: Express tagged versions to determine if increased levels affect autophagy markers

Autophagy Monitoring Techniques:

• Fluorescence microscopy: Using GFP-Atg8/LC3 to monitor autophagosome formation

• Western blotting: Analyzing Atg8/LC3 lipidation (LC3-I to LC3-II conversion)

• Transmission electron microscopy: For direct visualization of autophagic structures

• Flow cytometry: For quantitative analysis of autophagy markers

• De novo puncta tracking: As described in the research methodology

Specific Autophagy Induction Conditions:

• Mechanical stimulation:

  • Compression: Using the described apparatus for applying sustained compression

  • Fluid shear: Using flow chambers to apply defined shear stress

  • Stretch: Using elastic substrates

• Chemical induction:

  • 8Br-cGMP treatment: As described in the research (dose-responsive manner)

  • Amino acid starvation: Traditional autophagy induction method

  • Hyper-osmotic shock: As described in the research

• Combined stimuli: To identify potential synergistic or antagonistic effects

Protein Interaction Studies:

• Co-immunoprecipitation: With known autophagy proteins

• Proximity labeling: To identify proteins in close proximity during autophagy induction

• Subcellular localization: To determine if DDB_G0286829 relocates during autophagy

These approaches can establish whether DDB_G0286829 is a component of the autophagy machinery, a regulator of the process, or a substrate degraded by autophagy.

What experimental design is recommended for validating protein-protein interactions involving DDB_G0286829?

Validating protein-protein interactions for an uncharacterized protein requires a multi-layered approach. For DDB_G0286829, the following experimental design is recommended:

Stage 1: Identification of Potential Interaction Partners

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

  • Express His-tagged DDB_G0286829 in Dictyostelium

  • Perform pull-downs under various conditions (basal, starvation, cGMP stimulation, mechanical stress)

  • Identify co-purifying proteins by mass spectrometry

  • Use appropriate controls (empty vector, irrelevant His-tagged protein)

• Proximity-Based Labeling:

  • Generate BioID or APEX2 fusion with DDB_G0286829

  • Express in Dictyostelium and activate labeling

  • Identify biotinylated proteins by streptavidin pull-down and mass spectrometry

• Yeast Two-Hybrid Screening:

  • Use DDB_G0286829 as bait against Dictyostelium cDNA library

  • Test against specific candidates from autophagy pathways

Stage 2: Validation of Identified Interactions

• Co-immunoprecipitation:

  • Reciprocal pull-downs with tagged proteins

  • Endogenous protein interactions with specific antibodies

  • Test under different conditions to identify regulated interactions

• Fluorescence Resonance Energy Transfer (FRET):

  • Generate fluorescent protein fusions (e.g., CFP-DDB_G0286829 and YFP-Interactor)

  • Measure energy transfer in living cells

  • Map interaction domains through truncation mutants

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein fragments fused to potential interaction partners

  • Visualize interaction through reconstituted fluorescence

  • Determine subcellular localization of interactions

Stage 3: Functional Validation

• Genetic Interaction Studies:

  • Generate single and double knockouts

  • Compare phenotypes to identify epistatic relationships

  • Rescue experiments with wild-type and mutant variants

• Domain Mapping and Mutagenesis:

  • Create truncations to identify interaction domains

  • Perform site-directed mutagenesis of key residues

  • Test effect on binding and function

• Competitive Binding Assays:

  • Use peptides or domain fragments to disrupt interactions

  • Measure functional consequences of disruption

Stage 4: Structural Characterization

• Crosslinking Mass Spectrometry:

  • Identify specific residues involved in interactions

  • Map to structural models of the proteins

• Co-crystallization or Cryo-EM:

  • Determine structure of the protein complex

  • Identify detailed molecular interactions

This comprehensive approach provides multiple layers of evidence to confidently establish and characterize protein-protein interactions involving DDB_G0286829.

How can DDB_G0286829 research contribute to understanding protein quality control mechanisms?

Research on DDB_G0286829 may provide valuable insights into protein quality control mechanisms, particularly given Dictyostelium's exceptional ability to resist protein aggregation :

Potential Contributions:

• Novel Protein Quality Control Factors: If DDB_G0286829 participates in protein quality control, it may represent a previously uncharacterized component of these pathways. Dictyostelium has evolved mechanisms to maintain solubility of proteins with extended polyglutamine tracts that typically aggregate in other organisms . DDB_G0286829 could be part of this specialized machinery.

• Stress Response Integration: The protein's appearance in phosphoproteomic studies related to mechanical stress and cGMP signaling suggests potential roles in linking environmental stressors to proteostasis responses . This could reveal how cells coordinate protein quality control with other cellular processes.

• Evolutionary Insights: Comparative studies between Dictyostelium's protein quality control systems and those of other organisms could reveal evolutionary adaptations that enhance proteostasis. DDB_G0286829 may represent a Dictyostelium-specific innovation.

• Therapeutic Implications: Understanding how Dictyostelium prevents aggregation of polyglutamine-expanded proteins could inform therapeutic approaches for neurodegenerative diseases characterized by protein aggregation, such as Huntington's disease .

Research Approaches:

• Test whether DDB_G0286829 knockout affects the solubility of aggregation-prone proteins

• Investigate interactions between DDB_G0286829 and known quality control components (chaperones, degradation machinery)

• Examine expression changes in response to proteotoxic stressors

• Test if DDB_G0286829 expression in other cell types can confer aggregation resistance

• Identify potential human homologs or functional equivalents

This research direction could not only characterize DDB_G0286829 but also uncover novel principles of protein quality control with broad implications.

What is the significance of studying uncharacterized proteins like DDB_G0286829 in model organisms?

Studying uncharacterized proteins like DDB_G0286829 in model organisms such as Dictyostelium discoideum holds significant scientific value:

Scientific Significance:

• Filling Knowledge Gaps: Despite advances in genomics, a substantial portion of predicted proteins remains uncharacterized. Studies on DDB_G0286829 contribute to reducing this knowledge gap and completing our understanding of cellular systems.

• Discovering Novel Functions: Uncharacterized proteins often represent undiscovered biological processes or regulatory mechanisms. DDB_G0286829 may possess functions not previously described in biological systems.

• Evolutionary Insights: Comparing uncharacterized proteins across species can reveal evolutionary innovations and adaptations. Dictyostelium's position as a social amoeba provides a unique evolutionary perspective between unicellular and multicellular organisms.

• Systems Biology Completion: Complete understanding of cellular pathways requires characterization of all components. DDB_G0286829 may represent a missing piece in known signaling networks or cellular processes.

Methodological Advantages of Dictyostelium:

• Genetic Tractability: Dictyostelium is amenable to genetic manipulation, allowing straightforward generation of knockouts and transgenic lines.

• Cellular Simplicity with Multicellular Aspects: As a social amoeba, Dictyostelium offers insights into both unicellular processes and multicellular coordination.

• Unique Properties: Dictyostelium's resistance to protein aggregation and responsiveness to mechanical stimulation provide valuable experimental contexts for protein characterization.

• Conservation of Core Processes: Many fundamental cellular processes are conserved between Dictyostelium and higher eukaryotes, making findings potentially translatable.

Studying DDB_G0286829 not only characterizes this specific protein but contributes to broader understanding of proteome function and evolution, potentially revealing novel biology with implications beyond Dictyostelium.

How might research on DDB_G0286829 inform understanding of mechanically induced autophagy?

Research on DDB_G0286829 could significantly advance understanding of mechanically induced autophagy, a process with implications for multiple biological contexts:

Potential Insights:

• Signal Transduction Pathways: If DDB_G0286829 is phosphorylated in response to mechanical stimulation, it may represent a component of the signal transduction pathway linking mechanical forces to autophagy induction. Research has shown that sustained compression stimulates cyclic GMP (cGMP) production, which in turn induces autophagy . DDB_G0286829 could function within this pathway or in parallel mechanisms.

• Non-Canonical Autophagy Regulation: The research context indicates that mechanically induced autophagy may occur via non-canonical mechanisms, as "involvement of established autophagy signalling proteins was ruled out" . DDB_G0286829 could be part of this alternative regulatory mechanism.

• Integration of Multiple Stressors: DDB_G0286829 was identified in the context of both mechanical stress and hyper-osmotic stress-induced autophagy . It may therefore represent a convergence point for different cellular stressors.

• Novel Autophagy Components: As an uncharacterized protein, DDB_G0286829 could represent a previously unknown component of the autophagy machinery specific to mechanically induced autophagy.

Research Applications:

• Mechanobiology: Understanding how cells sense and respond to mechanical forces through autophagy has implications for tissues regularly subjected to mechanical stress (bone, muscle, cardiovascular system).

• Disease Relevance: Dysregulation of mechanosensing and autophagy is implicated in multiple diseases, including cancer, cardiovascular disease, and musculoskeletal disorders.

• Therapeutic Targeting: Identifying specific components of mechanically induced autophagy could provide novel therapeutic targets for diseases where this process is dysregulated.

• Biotechnology Applications: Understanding how mechanical forces regulate cellular processes could inform bioreactor design and tissue engineering approaches.

Research on DDB_G0286829 thus connects fundamental cell biology to applied biomedical science through the lens of mechanically induced autophagy.

What are the key research priorities for further characterization of DDB_G0286829?

Based on the available information and research context, the following priorities should guide further characterization of DDB_G0286829:

• Functional Characterization:

  • Generate knockout and overexpression models to identify phenotypes

  • Test for roles in autophagy, particularly under mechanical stimulation and osmotic stress

  • Investigate potential contributions to protein aggregation resistance

  • Examine developmental regulation and expression patterns

• Structural Studies:

  • Determine three-dimensional structure through X-ray crystallography or Cryo-EM

  • Identify functional domains and critical residues

  • Characterize the significance of the repetitive asparagine-rich regions

  • Investigate potential membrane association suggested by C-terminal hydrophobic residues

• Interaction Network Mapping:

  • Identify protein interaction partners under different conditions

  • Map phosphorylation sites and responsible kinases

  • Determine subcellular localization and potential translocation

  • Investigate incorporation into protein complexes

• Evolutionary Analysis:

  • Search for homologs or functional equivalents in other species

  • Examine conservation patterns across Dictyostelium species

  • Investigate selective pressures on the gene sequence

  • Compare with other uncharacterized proteins in Dictyostelium

• Integration with Known Pathways:

  • Determine relationship to cGMP signaling pathways

  • Investigate connections to established autophagy machinery

  • Examine links to protein quality control systems

  • Explore potential roles in mechanosensing

These research priorities would provide complementary insights into DDB_G0286829 function and significance, potentially revealing novel biology with broader implications beyond this specific protein.

How should researchers approach collaborative studies involving DDB_G0286829?

Collaborative studies on DDB_G0286829 should be structured to leverage diverse expertise and methodologies, maximizing research impact:

Collaboration Framework:

• Multidisciplinary Team Assembly:

  • Cell biologists specializing in Dictyostelium and autophagy

  • Structural biologists for protein characterization

  • Bioinformaticians for sequence and evolutionary analysis

  • Mechanobiologists for mechanical stimulation experiments

  • Mass spectrometry experts for phosphoproteomic and interaction studies

  • Neurodegenerative disease researchers interested in protein aggregation resistance

• Technology and Resource Sharing:

  • Share generated cell lines and constructs

  • Develop standardized protocols for DDB_G0286829 handling

  • Create a centralized database for experimental results

  • Establish common experimental conditions for cross-laboratory validation

• Coordinated Research Plan:

  • Divide research questions among laboratories based on expertise

  • Establish regular data sharing and discussion forums

  • Design experiments with cross-validation in different laboratories

  • Develop integrated publication strategies

• Translational Considerations:

  • Partner with biomedical researchers to explore disease relevance

  • Collaborate with computational biologists for systems-level integration

  • Engage with industry partners for potential biotechnological applications

  • Connect with clinicians if human homologs show disease associations

• Open Science Approach:

  • Share protocols, data, and resources openly

  • Preprint findings to accelerate knowledge dissemination

  • Consider collaborative funding mechanisms

  • Develop shared research tools and resources

This collaborative framework ensures comprehensive characterization of DDB_G0286829 while maximizing resource efficiency and accelerating discovery timelines.