SCP2D1 Human

What is SCP2D1 and how does it relate to the SCP protein family?

SCP2D1 (also known as C20orf79) is a human gene that encodes a protein belonging to the sterol carrier protein family. While SCP2D1 shares domain similarities with SCP2, they have distinct functions in lipid metabolism and transport. SCP2D1 contains a coding sequence of 471 base pairs and is expressed as a protein that can be studied using bacterial expression systems with His-GST tags . The better-characterized SCP2 family member functions in the transport of lipid hydroperoxides to mitochondria and plays a critical role in ferroptosis pathways, particularly in osteoarthritis pathology .

What is the genomic organization of human SCP2D1?

The human SCP2D1 gene is located on chromosome 20 (hence its alternative name C20orf79) . Research methodologies to study its genomic organization include:

• Restriction analysis using 5-NheI and 3-XhoI sites that flank the gene in expression constructs

• Sequencing verification using GST Forward primer (5'-CACGTTTGGTGGTGGCGAC-3') and T7 terminator primer (5'-GCTAGTTATTGCTCAGCGG-3')

• Comparative genomic analysis with related family members like SCP2, which is located on chromosome 1

What are the optimal conditions for expressing recombinant SCP2D1 protein?

For successful expression of recombinant SCP2D1 protein, researchers should follow these methodological guidelines:

• Vector selection: Use low-medium copy number bacterial expression vectors with the T7 promoter, such as pPB-His-GST

• Host selection: Choose E. coli strains that are DE3 lysogens, which provide a source of T7 RNA polymerase

• Induction parameters:

  • Culture density: Initiate induction at OD600 of 0.6-1.2

  • IPTG concentration: 0.05-1mM (requires empirical optimization)

  • Temperature and duration: These parameters must be optimized case-by-case

How can researchers purify SCP2D1 protein using the dual-tag system?

The SCP2D1 construct described in the literature contains a dual N-terminal tag system with 6X Histidine followed by Glutathione-S-Transferase (His-GST) . This design enables a two-step purification strategy:

• Primary capture: Immobilized metal affinity chromatography (IMAC) using the His-tag

• Secondary purification: Glutathione affinity chromatography using the GST portion

• Tag removal: The fusion tag is cleavable with TEV protease (the tag size is 27.9 kDa)

• Final polishing: Size exclusion chromatography to achieve high purity

This methodology allows for stringent purification under native conditions while preserving protein activity.

What role might SCP2D1 play in peroxisomal metabolism based on SCP2 family functions?

SCP2 is known to be involved in peroxisomal β-oxidation, with SCPx (encoded by the SCP2 gene) catalyzing the final step of this pathway . This process is critical for:

• Detoxification of very long-chain and branched-chain fatty acids

• Metabolism of cholesterol to form bile acids

Experimental approaches to investigate potential peroxisomal functions of SCP2D1 include:

• Subcellular fractionation to determine peroxisomal association

• Metabolomic profiling in cells with SCP2D1 knockdown/overexpression

• Analysis of peroxisomal marker proteins and metabolites in response to SCP2D1 manipulation

Could mutations in SCP2D1 contribute to metabolic or neurological disorders?

While specific SCP2D1 mutations have not been extensively characterized in the literature, insights from SCP2 mutations provide valuable context:

• SCPx deficiency resulting from SCP2 mutations has been associated with:

  • Progressive brainstem neurodegeneration

  • Cardiac dysrhythmia

  • Muscle wasting

  • Azoospermia

• A heterozygous SCP2 variant (c.572A>G, p.His191Arg) has been identified in a patient presenting with:

  • Abnormal plasma fatty acid profiles

  • Alterations in medium-, long-, and very long-chain fatty acids

Researchers investigating potential SCP2D1-related disorders should consider:

• Whole-genome or exome sequencing in patients with unexplained lipid metabolism disorders

• Functional characterization of identified variants using in vitro expression systems

• Generation of cellular and animal models expressing SCP2D1 variants

How might SCP2D1 be involved in inflammatory or degenerative conditions?

SCP2 has been implicated in osteoarthritis (OA) pathology, with high expression in human OA cartilage accompanied by ferroptosis hallmarks . Experimental evidence shows that:

• SCP2 promotes the accumulation of lipid hydroperoxides

• Inhibition of SCP2 protects mitochondria and reduces LPO levels

• This protection attenuates chondrocyte ferroptosis and alleviates OA progression in rat models

To investigate potential SCP2D1 involvement in similar conditions, researchers should:

• Compare SCP2D1 expression levels in normal versus diseased tissues

• Determine if SCP2D1 inhibition provides similar protective effects

• Identify specific inflammatory signaling pathways that may be affected by SCP2D1 function

What high-throughput methods can be used to identify SCP2D1 interaction partners?

Identifying protein-protein and protein-lipid interactions is crucial for understanding SCP2D1 function. Advanced methodologies include:

• Proximity-based labeling techniques:

  • BioID or TurboID fusion proteins to identify proximal interacting partners

  • APEX2 labeling for subcellular compartment-specific interactions

• Mass spectrometry-based approaches:

  • Immunoprecipitation coupled with LC-MS/MS

  • Crosslinking mass spectrometry (XL-MS) to capture transient interactions

  • Lipidomic analysis to identify bound lipid species

• Functional genomic screens:

  • CRISPR-Cas9 screens to identify genetic interactions

  • Synthetic lethal approaches to discover context-dependent functions

How can researchers differentiate between direct and indirect effects of SCP2D1 perturbation?

When analyzing phenotypes resulting from SCP2D1 manipulation, differentiating direct from indirect effects requires rigorous experimental design:

• Temporal analysis:

  • Use inducible expression/repression systems to track immediate versus delayed effects

  • Time-course experiments to establish causality chains

• Rescue experiments:

  • Structure-function analysis using domain mutants

  • Complementation with related family members like SCP2

• Direct target validation:

  • For transcriptional effects, use ChIP-seq or CUT&RUN

  • For metabolic effects, use isotope tracing to follow specific pathways

• Systems biology approaches:

  • Network analysis to position SCP2D1 in relevant pathways

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

What approaches could be used to modulate SCP2D1 activity for therapeutic purposes?

Based on findings from SCP2 research, several strategies could be explored for modulating SCP2D1 activity:

• Small molecule inhibitors:

  • Structure-based drug design targeting the lipid-binding domain

  • High-throughput screening of compound libraries

• Protein-level interventions:

  • Research on SCP2 has shown that fenofibrate and 4-hydroxytamoxifen can increase SCPx levels

  • Similar approaches could be tested for SCP2D1

• Gene therapy approaches:

  • AAV-mediated delivery for tissue-specific expression

  • CRISPR-based gene editing for correcting pathogenic variants

How might targeting SCP2D1 affect broader lipid metabolism pathways?

Therapeutic targeting of SCP2D1 would likely have downstream effects on multiple lipid metabolism pathways. Research on SCP2 indicates involvement in:

• PPAR signaling

• Serotonergic signaling

• Steroid biosynthesis

• Fatty acid degradation

Comprehensive metabolic profiling would be essential to understand the full impact of SCP2D1 modulation, particularly focusing on:

• Primary bile acid biosynthesis

• Linoleic acid metabolism

• Changes in free fatty acids, acylcarnitines, sterols, phospholipids, and sphingolipids

What are the most pressing unanswered questions about SCP2D1 function?

Several critical knowledge gaps remain in our understanding of SCP2D1:

• Tissue-specific expression patterns and subcellular localization

• Precise lipid binding specificity and transport mechanisms

• Physiological regulation of SCP2D1 expression

• Role in normal development and potential involvement in disease states

• Evolutionary conservation and divergence from other SCP family members

How might emerging technologies advance SCP2D1 research?

Novel technologies that could significantly advance SCP2D1 research include:

• Cryo-electron microscopy for high-resolution structural studies

• Single-cell multi-omics to understand cellular heterogeneity in SCP2D1 function

• Organoid models to study SCP2D1 in physiologically relevant 3D tissues

• Live-cell imaging with advanced probes to track lipid transport dynamics

• AI-driven prediction of protein-lipid interactions and drug discovery approaches