Recombinant Pan troglodytes Reticulon-1 (RTN1)

What is the molecular structure of Pan troglodytes RTN1 and how does it compare to human RTN1?

RTN1 is an endoplasmic reticulum (ER)-resident protein that belongs to the reticulon family. The protein contains an N-terminal domain and a reticulon homology domain with hydrophobic segments that integrate into the ER membrane. Pan troglodytes (chimpanzee) RTN1 shares high sequence homology with human RTN1, making it valuable for comparative studies of protein function. RTN1A, a major isoform, contains an extended N-terminal domain that distinguishes it from other isoforms such as RTN1C, which lacks most of the N-terminal domain and does not participate in ER stress response .

What are the primary cellular functions of RTN1 based on current research?

Research demonstrates that RTN1, particularly the RTN1A isoform, has several key functions:

• Shaping and curvature of ER membranes

• Regulation of ER-mitochondria contacts (EMCs)

• Modulation of ER stress response in tubular epithelial cells

• Participation in mitochondrial homeostasis and function

• Involvement in cellular apoptotic and inflammasome pathways

• Potential role in kidney tubular epithelial cell (TEC) injury in diabetic kidney disease (DKD)

How can researchers differentiate between the functional roles of different RTN1 isoforms?

To distinguish between RTN1 isoform functions, researchers should:

• Design experiments comparing full-length RTN1A with RTN1C, which lacks most of the N-terminal domain

• Use co-immunoprecipitation followed by mass spectrometry to identify isoform-specific protein interactions

• Conduct comparative functional assays, as RTN1A participates in ER stress response while RTN1C does not

• Create isoform-specific overexpression and knockdown models to evaluate differential effects on cellular phenotypes

• Analyze tissue-specific expression patterns of different isoforms

Evidence shows that proteins co-immunoprecipitating with RTN1A, but not RTN1C, are predominantly mitochondrial proteins, suggesting isoform-specific roles in ER-mitochondrial crosstalk .

What expression systems yield optimal results for producing functional recombinant Pan troglodytes RTN1?

When expressing recombinant Pan troglodytes RTN1:

• Mammalian expression systems (HEK293, CHO cells) are preferred for maintaining proper post-translational modifications

• For tetracycline-inducible expression, systems like the Pax8-rtTA;tetO-RTN1A can achieve controlled expression in specific cell types

• Bacterial artificial chromosome (BAC) systems can facilitate genetic manipulation, similar to the approach used for chimpanzee adenovirus vectors

• Consider using codon-optimized sequences for the expression system of choice

• Include appropriate epitope tags (FLAG, His) for detection and purification while ensuring tags don't interfere with protein function

• For membrane proteins like RTN1, detergent optimization is critical during extraction and purification

What are methodologically sound approaches to study RTN1's role in ER-mitochondrial contacts?

To investigate RTN1's role in ER-mitochondrial contacts:

• In situ proximity ligation assay (PLA): Use antibodies against RTN1 and mitochondrial markers to visualize and quantify protein interactions at the ER-mitochondria interface

• Electron microscopy: Measure the distance between ER and mitochondria (optimal range 10-30nm) in cells with varying levels of RTN1 expression

• Live-cell imaging: Track fluorescently tagged RTN1 and organelle markers to observe dynamic interactions

• Co-immunoprecipitation: Identify RTN1-interacting proteins, particularly mitochondrial proteins like hexokinase-1 and VDAC1

• Quantitative image analysis: Measure co-localization of RTN1 with ER-mitochondria contact site markers

Research demonstrates that RTN1A overexpression decreases the average distance between ER and mitochondria, suggesting increased interaction between these organelles .

What experimental controls are essential when evaluating RTN1 expression levels in disease models?

When studying RTN1 in disease models, include these critical controls:

• Matched tissue/cell samples from healthy and diseased subjects

• Comparison of multiple isoforms (RTN1A vs RTN1C) to determine isoform-specific effects

• Quantification of both mRNA (qPCR) and protein levels (Western blot, immunofluorescence)

• Proper housekeeping genes or loading controls appropriate for the disease model

• Correlation analysis with clinical parameters (e.g., estimated glomerular filtration rate in kidney disease)

• Inclusion of both global and tissue-specific transgenic models when possible

• Time-course analyses to track expression changes during disease progression

Research shows RTN1A expression is markedly increased in tubular epithelial cells of diseased kidneys and inversely correlates with renal function in diabetic patients .

How can researchers effectively design transgenic models to study tissue-specific RTN1 functions?

For development of tissue-specific RTN1 transgenic models:

• Selection of appropriate promoter systems:

  • Use tetracycline-inducible systems (e.g., Pax8-rtTA for tubular epithelial cells)

  • Consider Cre-loxP recombination for conditional expression

  • Ensure tissue specificity through immunostaining validation with tissue markers

• Transgene design considerations:

  • Include species-specific RTN1 sequences (human or chimpanzee)

  • Optimize expression levels (a 4-fold increase has shown phenotypic effects)

  • Consider co-expression of fluorescent reporters for tracking

• Validation approaches:

  • Confirm transgene expression using qPCR for both endogenous and transgenic transcripts

  • Validate protein expression using immunostaining and co-localization with tissue markers

  • Test inducibility using doxycycline administration (e.g., 625mg/kg chow for 3 weeks)

• Phenotypic characterization:

  • Compare tissue-specific versus global expression/knockdown models

  • Examine both baseline and disease-challenged conditions

  • Analyze tissue-specific and systemic parameters

Studies demonstrate that Pax8-rtTA;tetO-RTN1A transgenic mice with doxycycline induction achieved a 4-fold increase in RTN1A expression specifically in tubular cells .

What methodologies can detect and quantify RTN1-mediated ER stress response in experimental models?

To evaluate RTN1-mediated ER stress:

• Molecular markers:

  • Measure expression of ER stress markers (GRP78/BiP, CHOP, XBP1 splicing, ATF4, ATF6)

  • Quantify phosphorylation status of PERK and eIF2α

  • Assess ER-associated degradation pathway components

• Imaging approaches:

  • Immunofluorescence co-localization of RTN1 with ER stress markers

  • Ultrastructural analysis of ER morphology using electron microscopy

• Functional assays:

  • ER calcium measurements using fluorescent indicators

  • Protein folding capacity assays

  • Unfolded protein response reporter systems

• Pharmacological interventions:

  • ER stress inducers (tunicamycin, thapsigargin) as positive controls

  • ER stress mitigators (chemical chaperones like 4-PBA) for rescue experiments

  • Compare responses in RTN1 wildtype vs. modified models

• In vivo assessment:

  • Tissue-specific analysis of ER stress markers in transgenic models

  • Comparison between healthy and disease states (e.g., diabetes)

  • Temporal analysis during disease progression

Research demonstrates that increased RTN1A expression exacerbates the ER stress response to promote kidney disease progression .

How can researchers effectively assess the impact of RTN1 on mitochondrial function?

To evaluate RTN1's effects on mitochondrial function:

• Mitochondrial respiration analysis:

  • Measure oxygen consumption rate using Seahorse XF analyzer

  • Assess basal, maximal, and spare respiratory capacity

  • Quantify ATP production capacity

• Mitochondrial membrane potential:

  • Use fluorescent indicators (TMRM, JC-1) to measure membrane potential

  • Assess potential changes in response to RTN1 modulation

• ROS production:

  • Measure mitochondrial ROS with specific indicators

  • Assess oxidative stress markers in tissues/cells

• Mitochondrial dynamics:

  • Analyze mitochondrial morphology and network structure

  • Quantify fission/fusion protein levels and activity

• Mechanistic protein interaction studies:

  • Investigate RTN1 interaction with mitochondrial proteins (hexokinase-1, VDAC1)

  • Assess impact on protein-protein interactions among mitochondrial components

  • Identify disruption of normal mitochondrial protein complexes

Research shows RTN1A interacts with mitochondrial hexokinase-1 and VDAC1, interfering with their association and leading to activation of apoptotic and inflammasome pathways .

How can comparative analysis between human and Pan troglodytes RTN1 advance understanding of therapeutic targets?

Comparative analysis offers several research advantages:

• Evolutionary conservation analysis:

  • Identify highly conserved domains between species, suggesting functional importance

  • Compare with other primate RTN1 sequences to determine primate-specific regions

  • Analyze species-specific variations that may correlate with differential disease susceptibility

• Structural-functional relationships:

  • Use sequence variations to inform structure-function relationships

  • Employ homology modeling to predict functional consequences of species differences

  • Design chimeric constructs to map species-specific functional domains

• Disease modeling considerations:

  • Leverage lower human seroprevalence of chimpanzee-derived vectors for potential therapeutic delivery

  • Compare species-specific interactions with cellular machinery

  • Identify potential species-specific post-translational modifications

• Therapeutic development approaches:

  • Target conserved domains for broad-spectrum interventions

  • Exploit species differences to develop specific inhibitors

  • Use chimpanzee adenovirus vectors with low human seroprevalence for potential RTN1-targeting gene therapy

Research on chimpanzee adenovirus vectors shows particularly low human seroprevalence for some serotypes, which could inform vector selection for potential RTN1-targeting therapeutics .

What research design best addresses the differentiation between RTN1's roles in glomerular versus tubular pathology in kidney disease?

To distinguish RTN1's compartment-specific roles:

• Cell-type specific models:

  • Generate and compare podocyte-specific versus tubular-specific RTN1 transgenic models

  • Utilize conditional knockout models targeting specific kidney compartments

  • Employ cell-type specific promoters (e.g., Pax8 for TEC, podocin for podocytes)

• Comprehensive phenotyping approach:

  • Assess glomerular parameters: albuminuria, glomerular histology, podocyte markers

  • Measure tubular parameters: KIM1 expression, tubulointerstitial fibrosis, tubular injury markers

  • Evaluate systemic parameters: blood urea nitrogen, serum creatinine, estimated GFR

• Molecular profiling strategies:

  • Perform laser capture microdissection to isolate specific kidney compartments

  • Conduct single-cell RNA sequencing to identify cell-specific RTN1 expression patterns

  • Compare proteomics of isolated glomeruli versus tubular fractions

• Translational relevance analysis:

  • Correlate findings with clinical observations of DKD phenotypes with/without albuminuria

  • Identify biomarkers specific to RTN1-mediated tubular versus glomerular pathology

  • Develop targeted intervention strategies for specific kidney compartments

Studies reveal that TEC-specific RTN1A overexpression worsened tubulointerstitial fibrosis and kidney function without significantly affecting glomerular injury or albuminuria, mimicking progressive diabetic kidney disease without overt proteinuria .

What methodological considerations are essential when investigating RTN1's role in ER-mitochondrial crosstalk in disease settings?

For investigating RTN1 in ER-mitochondrial crosstalk:

• Model system selection:

  • Compare immortalized cell lines versus primary cells

  • Evaluate acute versus chronic disease models

  • Consider 2D versus 3D culture systems or organoids

• Molecular mechanisms assessment:

  • Identify key interacting proteins at ER-mitochondrial contact sites

  • Map protein domains responsible for interactions

  • Generate interaction-deficient mutants for functional validation

• Subcellular fractionation approach:

  • Isolate mitochondria-associated ER membranes (MAMs)

  • Compare protein composition of MAMs in normal versus disease states

  • Assess RTN1 enrichment in different subcellular compartments

• Dynamic interaction analysis:

  • Employ live-cell imaging with optogenetic tools

  • Use FRET/BRET techniques to measure protein-protein interactions

  • Quantify effects of disease-relevant stressors on interaction dynamics

• Translational opportunities:

  • Design peptides or small molecules targeting RTN1-specific interactions

  • Evaluate existing drugs that modulate ER-mitochondrial communication

  • Develop biomarkers of disrupted ER-mitochondrial contacts

Research demonstrates that RTN1A is a component of ER-mitochondrial contacts, and its overexpression decreases the average distance between ER and mitochondria in diabetic models, promoting disease progression through enhanced ER-mitochondrial crosstalk .

What are the major technical challenges in expressing and purifying functional recombinant membrane proteins like RTN1?

Membrane protein expression challenges and solutions:

Researchers should systematically optimize these parameters while monitoring protein quality at each step.

How can researchers address contradictory findings between in vitro and in vivo RTN1 studies?

To reconcile contradictory findings:

• Systematic comparison analysis:

  • Directly compare protein expression levels between systems

  • Assess differences in post-translational modifications

  • Evaluate cellular context variations (interactions, compartmentalization)

• Dosage effect evaluation:

  • Test multiple expression levels (low, moderate, high)

  • Compare acute versus chronic expression models

  • Determine threshold levels for phenotypic effects (4-fold increase shows effects in vivo)

• Context-dependent function analysis:

  • Evaluate effects under basal versus stressed conditions

  • Compare healthy versus disease backgrounds

  • Assess species-specific differences in regulatory networks

• Technical validation approach:

  • Employ multiple independent techniques to measure same endpoints

  • Use different model systems to confirm key findings

  • Validate antibody and reagent specificity across systems

Studies show that RTN1A knockdown attenuated kidney injury in vivo, while RTN1A overexpression worsened diabetic kidney phenotypes, demonstrating consistent direction of effect across models .

What genetic and evolutionary considerations should inform experimental design when working with Pan troglodytes RTN1?

Important genetic and evolutionary considerations:

• Genomic sequence analysis:

  • Analyze conserved versus divergent regions between human and chimpanzee RTN1

  • Identify primate-specific regulatory elements affecting expression

  • Consider chimpanzee-specific LINE-1 elements that may affect nearby gene regulation

• Expression pattern evaluation:

  • Compare tissue-specific expression patterns between species

  • Assess isoform distribution differences

  • Evaluate regulatory mechanisms controlling expression

• Functional conservation testing:

  • Determine if chimpanzee RTN1 can functionally replace human RTN1

  • Identify species-specific interaction partners

  • Test response to various cellular stressors across species

• Genome editing considerations:

  • Design species-specific guide RNAs for CRISPR/Cas9 applications

  • Consider potential off-target effects unique to each genome

  • Employ appropriate genomic reference databases (e.g., panTro5)

Research shows significant genomic differences between human and chimpanzee, including chimpanzee-specific L1 elements that have contributed to genome diversity and variations during primate evolution .