Recombinant Pongo abelii Serine palmitoyltransferase 1 (SPTLC1)

What is Pongo abelii Serine palmitoyltransferase 1 and what is its biological function?

Pongo abelii Serine palmitoyltransferase 1 (SPTLC1) is a protein originally isolated from Sumatran orangutan (Pongo abelii) that functions as a critical component of the serine palmitoyltransferase enzyme complex. This protein has the UniProt accession number Q5R9T5 and spans 473 amino acids in its full-length form . Structurally, SPTLC1 lacks an intrinsic catalytic site but serves an essential membrane tethering role required for the enzymatic activity of the SPTLC1/SPTLC2 heterocomplex . The protein is officially classified with the EC number 2.3.1.50 and is involved in sphingolipid biosynthesis, specifically catalyzing the condensation of serine with palmitoyl-CoA to form 3-ketodihydrosphingosine . Recent studies have expanded our understanding of SPTLC1's biological roles beyond its enzymatic function, revealing its involvement in cellular stress responses and possible interactions with multiple regulatory proteins, including ABCA1 .

What are the alternative nomenclature and structural features of SPTLC1?

Researchers should be aware of several alternative names when searching literature about this protein. SPTLC1 is also known as Long chain base biosynthesis protein 1 (LCB 1), Serine-palmitoyl-CoA transferase 1 (SPT 1 or SPT1) . The complete amino acid sequence of Pongo abelii SPTLC1 starts with MATATEQ and ends with EVAQAVLL, with a full sequence of 473 amino acids that includes multiple functional domains responsible for its biological activities . The protein contains regions essential for membrane association, partner protein binding, and participation in the sphingolipid synthesis pathway. Mutations in the human SPTLC1 gene have been linked to hereditary sensory neuropathy (HSN), highlighting its clinical significance beyond basic cellular functions . Understanding these structural features is critical when designing experiments involving protein modification, such as creating tagged versions for localization studies or functional analyses.

How is recombinant SPTLC1 properly stored and handled in laboratory settings?

Proper storage and handling of recombinant SPTLC1 is crucial for maintaining protein stability and experimental reproducibility. The recommended storage condition for recombinant Pongo abelii SPTLC1 is at -20°C in a Tris-based buffer containing 50% glycerol, which has been optimized specifically for this protein . For extended storage periods, conserving the protein at -80°C can further preserve its integrity and activity . Working aliquots should be maintained at 4°C and used within one week to prevent degradation . Importantly, repeated freezing and thawing cycles should be avoided as they can compromise protein structure and function . When planning long-term studies, it is advisable to prepare multiple small aliquots during initial receipt of the protein to minimize freeze-thaw cycles. Researchers should also consider the specific buffer conditions required for their experimental systems, as buffer components may affect protein behavior in various assays.

What approaches are recommended for generating tagged SPTLC1 constructs for cellular studies?

Several validated approaches exist for generating tagged SPTLC1 constructs that maintain protein functionality while allowing detection and localization studies. For N-terminal GFP-tagged SPT1, researchers can amplify the SPTLC1 gene using specific primers (5'-ATGGCGACCGCCACGGAGCAGTGG-3' and 5'-GCCTAGAGCAGGACGGCCTGGGCT-3') and clone the PCR fragment into appropriate vectors such as pEGFP-C2 . For C-terminal GFP tagging, a slightly modified approach using primers 5'-ATGGCGACCGCCACGGAGCAGTGG-3' and 5'-GAGCAGGACGGCCTGGGCTACCTC-3' followed by subcloning into vectors like pEGFP-N1 has proven effective . When smaller epitope tags are preferred, techniques for creating small peptide-tagged SPT1 have been established using vectors such as pCMV-MAT-FLAG, with primers incorporating sequences for tags like HA . More sophisticated internal tagging approaches are also documented, where researchers amplify separate portions of the gene and use overlap extension techniques to insert tags at specific internal locations . The choice of tagging approach should be guided by the specific research question, as tag location can affect protein localization, interactions, and function.

What are the validated protocols for establishing stable SPTLC1 recombinant cell lines?

Establishing stable SPTLC1 recombinant cell lines requires careful consideration of cell type, transfection method, and selection strategy. A well-documented approach involves co-transfecting expression plasmids into appropriate cell lines (such as HEK293) using transfection reagents like Superfect . Following transfection, stable integrants can be selected by adding appropriate antibiotics to the culture media (e.g., 400 μg/ml geneticin or 200 μg/ml zeocin) 48 hours post-transfection . For co-transfection experiments, both antibiotics should be included in the selection media . The media should be changed approximately every 4 days during the selection process, which typically takes about 2 weeks . After selection, surviving colonies should be isolated and expanded in individual wells of multi-well plates for further characterization . Validation of recombinant expression should include both RT-PCR to confirm gene transcription and western blotting to verify protein expression levels . For transfection of difficult-to-transfect cell lines, lipid-based transfection methods have been successful, with protocols specifying the use of 1 μg of vector DNA per 10^5 cells growing near confluence in 6-well plates .

How should subcellular localization studies of SPTLC1 be performed and interpreted?

Subcellular localization studies of SPTLC1 require rigorous methodology to generate reliable results, especially given evidence that SPTLC1 can be found in multiple subcellular compartments . Confocal immunofluorescence microscopy represents a gold-standard approach for such analyses. A validated protocol involves growing cells on culture slides, fixing with 3% paraformaldehyde in PBS for 15 minutes, washing thoroughly, and permeabilizing with 0.1% Saponin . After blocking with 1% bovine serum albumin, cells should be incubated with primary antibodies at 1:500 dilution for 1 hour at room temperature, followed by fluorophore-conjugated secondary antibodies at 1:1000 dilution . F-actin cytoskeleton can be simultaneously visualized using Phalloidin 596 at 1:200 dilution . High-resolution imaging systems such as the Zeiss LSB 700 confocal microscope are recommended for optimal visualization . When interpreting results, researchers should be aware that SPTLC1 distribution patterns can change dramatically in response to cellular stressors or chemical treatments, with evidence showing rapid redistribution to focal adhesion sites in response to compounds like Celecoxib . Co-localization studies with known markers of subcellular compartments should be included to properly identify the precise location of SPTLC1 under various experimental conditions.

How does SPTLC1 expression influence cellular stress response mechanisms?

SPTLC1 has emerged as a stress-responsive protein with significant influence on cellular response to various stressors. Research indicates that recombinant cells expressing C-terminal modified SPTLC1 display altered sensitivity to cytotoxic compounds, including environmental contaminants like 3-methylcholanthrene and therapeutic agents . This modulation of stress response is thought to occur through SPTLC1's interaction with key regulatory proteins involved in cellular homeostasis . The protein's expression is naturally enhanced in rapidly proliferating normal cells undergoing morphological changes, including lung, stomach, and intestinal epithelium, as well as in stromal fibroblasts surrounding malignant tissues . When studying SPTLC1's role in stress response, researchers should design experiments that examine both immediate and long-term cellular outcomes following stressor exposure. Cell viability assays have demonstrated that SPTLC1 recombinant cells exhibit differential, dose-dependent resistance to excitotoxic levels of compounds like Glutamate . The protein's influence extends to transcriptional responses, with evidence showing SPTLC1 modulates Cyp450 transcriptional expression in cells treated with drugs like Celecoxib . These findings suggest SPTLC1 may function as a signaling node in stress response networks, making it a valuable target for studies on cellular adaptation to adverse conditions.

What protein-protein interactions involving SPTLC1 have been identified and how can they be studied?

An increasing number of SPTLC1 binding partners have been identified through antibody-mediated pull-down approaches and co-localization studies, revealing a complex interactome with significant biological implications . Notable among these interactions is SPTLC1's association with the 90 kiloDalton heat shock protein (Hsp90), a molecular chaperone critical for the functional activation of numerous signaling proteins involved in cellular homeostasis and stress response gene expression . Recent studies have also detected interaction between SPTLC1 and ABCA1, suggesting potential roles in membrane transport processes . To investigate these interactions, researchers should employ complementary approaches including co-immunoprecipitation, proximity ligation assays, and FRET-based techniques to verify direct physical associations. Tagged versions of SPTLC1 (as described in section 2.1) can facilitate these studies, though care must be taken to ensure tags do not interfere with the interactions being studied . The biological significance of these interactions should be assessed through functional assays, as the complexity of cellular morphological and behavioral changes associated with SPTLC1 gene mutations may be partly attributable to altered protein-protein interactions rather than simply changes in metabolic derivatives . The temporal-spatial dynamics of these interactions, particularly under various stress conditions, represent an important frontier in understanding SPTLC1's diverse cellular functions.

How does C-terminal modification of SPTLC1 alter cellular chemosensitivity and gene expression?

C-terminal modification of SPTLC1 has profound effects on cellular chemosensitivity and gene expression patterns, making it a significant consideration in experimental design. Using cell proliferation as an index of drug sensitivity, studies have demonstrated that recombinant SPTLC1 expression modulates dose-dependent sensitivity to various compounds . Cells transfected with C-terminal modified SPTLC1 recombinant vectors show differential sensitivity to chemotherapeutic agents compared to parental cell lines . For example, in experiments with Celecoxib (a COX-2 inhibitor with anti-neoplastic properties) combined with Geldanamycin, C-terminal modified SPTLC1 recombinant cells exhibited significantly higher sensitivity than parental or isogenic control lines . At the transcriptional level, SPTLC1 has been shown to modulate the expression of important metabolic genes, including Cyp450 family members in response to drug treatments . This modulation can be visualized and quantified through RT-PCR techniques using RNA harvested from treated and untreated cells . When designing experiments to investigate these effects, researchers should include appropriate controls and consider the cell-type specificity of responses, as different cancer cell lines (such as Glioma LN18, SKN-SH, PC3 prostate cancer, and 647V bladder cancer) have shown varying patterns of chemosensitivity modulation by SPTLC1 .

What controls should be included when studying SPTLC1 in cellular models?

When studying SPTLC1 in cellular models, a comprehensive set of controls is essential for generating reliable and interpretable data. For recombinant expression studies, researchers should include both parental (untransfected) cell lines and isogenic controls (cells transfected with empty vector or an irrelevant protein) to distinguish effects specific to SPTLC1 from those due to the transfection and selection process . When investigating SPTLC1's role in chemosensitivity, dose-response curves should be generated with multiple concentrations of test compounds, and cells should be assessed at several time points to capture both immediate and delayed effects . Co-treatment experiments, where cells are exposed to combinations of compounds (e.g., Celecoxib with Sulfasalazine, 3-methylcholanthrene, or Geldanamycin), require additional controls to account for potential drug interactions independent of SPTLC1 function . For localization studies, controls should include cells stained with secondary antibody alone to assess background fluorescence, as well as co-staining with markers of specific subcellular compartments to precisely define SPTLC1 distribution . When examining SPTLC1's effects on gene expression, housekeeping genes should be included for normalization, and temporal considerations are important as expression changes may vary over time following treatment .

How can SPTLC1 recombinant expression be validated and quantified?

Validation and quantification of SPTLC1 recombinant expression require multiple complementary approaches to ensure experimental rigor. Following transfection and selection of stable cell lines, RT-PCR should be performed to confirm transcription of the SPTLC1 gene, using primers specific to the introduced construct . Western blotting provides protein-level validation and should be conducted using antibodies specific to either SPTLC1 itself or to any epitope tags incorporated into the recombinant protein . For GFP-tagged constructs, direct fluorescence visualization can provide initial confirmation of expression and information about localization patterns . Quantitative PCR (qPCR) allows precise measurement of transcript levels and can be performed using RNA harvested from cells, with approximately 1 μg RNA reverse transcribed to generate cDNA for analysis . When comparing multiple recombinant lines, it is essential to establish relative expression levels, as differences in expression can influence experimental outcomes independently of the specific modifications being studied . For functional validation, researchers should determine whether the recombinant protein maintains expected activities and interactions, particularly when studying modified versions such as C-terminal altered SPTLC1 . The expression of recombinant SPTLC1 should be monitored periodically during long-term culture, as expression levels may change over time due to silencing or selection pressures.

What analytical approaches are recommended for studying SPTLC1's effect on cellular proliferation and viability?

When investigating SPTLC1's effects on cellular proliferation and viability, researchers should employ rigorous analytical approaches that capture both subtle and dramatic changes in cell behavior. Cell viability assays represent a fundamental tool, with data typically presented as relative cell viability compared to untreated controls for each cell line . These assays should be performed in 96-well culture plates with cells exposed to increasing concentrations of test compounds for standardized time periods (typically 48 hours) . For comparing chemosensitivity between parental cell lines and SPTLC1 recombinants, dose-response curves should be generated and analyzed using appropriate statistical methods to determine EC50 values and confidence intervals . Co-treatment experiments, where cells are exposed to combinations of compounds, require factorial experimental designs and appropriate controls to distinguish additive, synergistic, or antagonistic effects . Beyond simple viability measures, complementary assays examining cell cycle distribution, apoptosis markers, and morphological changes provide deeper insights into the mechanisms underlying SPTLC1's effects . For long-term effects, colony formation assays can reveal changes in anchorage-independent growth, a condition associated with increased COX-2 expression where SPTLC1 has been found to play a role . Statistical analysis should account for biological variability by including multiple biological replicates and appropriate statistical tests based on data distribution.

How does SPTLC1 contribute to inflammation-associated cellular processes?

Emerging evidence suggests significant involvement of SPTLC1 in inflammation-associated cellular processes, opening new research avenues for investigating this protein beyond its canonical role in sphingolipid metabolism. Studies in inflammation-associated cancer cell lines have revealed that SPTLC1 expression patterns influence cellular responses to anti-inflammatory compounds such as Celecoxib, a potent inhibitor of cyclooxygenase 2 (COX-2) . Confocal microscopy has shown that Celecoxib treatment mediates both rapid and enhanced redistribution of SPTLC1 and COX-2 to focal adhesion sites, suggesting potential functional interaction between these proteins in inflammatory signaling . SPTLC1 expression is also observed in cells growing as anchorage-independent colonies, a condition associated with increased COX-2 expression . Researchers investigating this area should design experiments that specifically examine the relationship between SPTLC1 and established inflammatory pathways, including the effects of SPTLC1 expression on pro-inflammatory cytokine production, NF-κB signaling, and inflammatory cell recruitment. The potential reciprocal regulation between SPTLC1 and inflammatory mediators represents a particularly promising area for investigation, with implications for understanding chronic inflammatory conditions and inflammation-associated malignancies.

What is known about SPTLC1 mutations and their effects on protein function?

Mutations in the SPTLC1 gene have been linked to hereditary sensory neuropathy disease (HSN), highlighting the clinical relevance of this protein beyond basic cellular functions . Research into how these mutations affect protein function has revealed complex consequences that extend beyond alterations in enzymatic activity. Studies suggest that the abnormal morphology and behavior of cultured cells following SPTLC1 perturbation may be attributable to both changes in metabolic derivatives and altered protein-protein interaction characteristics . When designing experiments to investigate mutation effects, researchers should consider both the direct impact on SPTLC1's role in sphingolipid biosynthesis and the potential disruption of its non-enzymatic functions in regulating important aspects of cell physiology, growth, inflammation, and stress response mechanisms . Comparative studies between wild-type and mutant SPTLC1 should examine subcellular localization patterns, protein-protein interaction profiles, and effects on downstream signaling pathways. Novel mutations should be characterized through structural modeling, in vitro enzyme activity assays, and cellular phenotyping to establish structure-function relationships. The temporal-spatial dynamics of mutant SPTLC1 under various cellular stressors may provide insights into the pathophysiology of associated neurological disorders.