Recombinant Human Probable low affinity copper uptake protein 2 (SLC31A2)

What is the molecular structure and basic properties of human SLC31A2?

SLC31A2 (human probable low affinity copper uptake protein 2) is a 143 amino acid protein encoded by the SLC31A2 gene. The gene consists of 4 exons with a protein-coding transcript of 1785 base pairs. The protein contains 3 transmembrane domains and is identified in UniProt by the accession number O15432. SLC31A2 is part of the SLC31 family of copper transporters that serve as major gateways for copper acquisition in eukaryotes .

The complete amino acid sequence is:
MAMHFIFSDTAVLLFDFWSVHSPAGMALSVLVLLLLAVLYEGIKVGKAKLLNQVLVNLPTSISQQTIAETDGDSAGSDSFPVGRTHHRWYLCHFGQSLIHVIQVVIGYFIMLAVMSYNTWIFLGVVLGSAVGYYLAYPLLSTA

The table below summarizes key properties of SLC31A2:

How does SLC31A2 expression vary across different tissues?

SLC31A2 is ubiquitously expressed across all human organs and tissues examined, though with notably higher expression levels in the liver and kidney . This broad expression pattern suggests a fundamental role in cellular function, potentially related to copper homeostasis across multiple tissue types. Unlike some tissue-specific copper transporters, SLC31A2's widespread presence indicates its involvement in basic cellular processes rather than specialized tissue functions.

The expression pattern differs from SLC31A1 (CTR1) in terms of both subcellular localization and tissue-specific regulatory patterns, though both show high expression in metabolically active organs like the liver and kidney . This expression pattern correlates with tissues that have high copper processing demands, suggesting potential roles in copper detoxification or recirculation.

What is the subcellular localization of SLC31A2 and how does it differ from CTR1?

Unlike SLC31A1 (CTR1), which primarily localizes to the plasma membrane for cellular copper uptake, SLC31A2 predominantly resides within intracellular compartments . Specifically, SLC31A2 is found in the membranes of intracellular organelles such as vacuoles, vesicles, endosomes, and lysosomes in mammalian cells.

This intracellular localization pattern is similar to several evolutionarily related proteins including budding yeast CTR2, fission yeast CTR6, and Arabidopsis thaliana COPT5 . The distinct localization pattern suggests that SLC31A2 likely functions in intracellular copper transport, possibly mediating copper movement between organelles or participating in copper recycling pathways rather than primary uptake from the extracellular environment.

What is the functional relationship between SLC31A2 and cisplatin resistance?

Research indicates a significant relationship between SLC31A2 and resistance to the platinum-based anticancer drug cisplatin. Interestingly, SLC31A2 expression has been associated with reduced cisplatin efficacy, as knock-down of SLC31A2 leads to enhanced cellular uptake of cisplatin . This relationship appears inversely related to that of CTR1, where decreased expression correlates with cisplatin resistance.

The mechanistic basis for this relationship likely stems from SLC31A2's role in intracellular compartmentalization of copper and potentially platinum compounds. Given that both copper and platinum are transition metals with similar chemical properties, the copper transport system can influence cisplatin accumulation and distribution . SLC31A2's predominant localization in intracellular vesicles and organelles may sequester cisplatin away from nuclear DNA, its primary therapeutic target, thereby reducing drug efficacy.

Understanding this relationship has significant implications for cancer treatment, as modulation of SLC31A2 expression or activity could potentially sensitize resistant tumors to platinum-based chemotherapies. Researchers exploring platinum drug resistance mechanisms should consider SLC31A2 expression levels as a potential biomarker for treatment response prediction.

How does SLC31A2 contribute to copper homeostasis compared to other copper transporters?

SLC31A2 likely contributes to intracellular copper redistribution rather than primary uptake, potentially mobilizing copper from lysosomal or endosomal compartments during specific cellular conditions. This function would complement the activity of other copper transporters like ATP7A and ATP7B, which move copper to the secretory pathway or mediate copper excretion.

In the broader context of copper homeostasis, SLC31A2 may provide a secondary copper acquisition pathway when primary mechanisms are compromised. Studies of CTR1-knockout embryos reveal that these organisms still maintain approximately one-third of normal copper levels through CTR1-independent copper transport systems . While not definitively identified as part of this backup system, SLC31A2 could potentially contribute to this alternative uptake pathway, though other transporters like DMT1 and ZIP proteins have also been implicated .

What is the role of SLC31A2 in cancer progression and its potential as a prognostic marker?

The prognostic value of SLC31A2 expression suggests it could be incorporated with other clinical parameters for improved patient risk stratification in ccRCC . Given the importance of copper in angiogenesis, tumor growth, and metastasis, SLC31A2's role in intracellular copper trafficking may influence multiple aspects of cancer progression beyond just survival metrics.

The relationship between SLC31A2 and cisplatin resistance adds another dimension to its relevance in cancer therapy. Tumors with high SLC31A2 expression may exhibit reduced response to platinum-based chemotherapies, potentially necessitating alternative treatment approaches or combination strategies that target SLC31A2-mediated resistance mechanisms.

What are the recommended methods for generating and validating recombinant SLC31A2 protein?

Recombinant expression of SLC31A2 presents challenges typical of multi-pass membrane proteins. For successful expression, researchers should consider the following methodological approaches:

• Expression System Selection: Mammalian expression systems (HEK293 or CHO cells) are preferable for maintaining proper folding and post-translational modifications of human SLC31A2. Alternatively, insect cell systems (Sf9 or High Five) can provide higher yields while maintaining eukaryotic processing capabilities.

• Construct Design:

  • Include a cleavable N-terminal signal peptide to ensure proper membrane targeting

  • Add epitope tags (His, FLAG, or Strep) preferably at the C-terminus to minimize interference with function

  • Consider fusion with GFP or other fluorescent proteins for localization studies

• Validation Methods:

  • Western blot analysis using either tag-specific or SLC31A2-specific antibodies

  • Immunofluorescence to confirm expected intracellular localization

  • Functional assays measuring copper transport capabilities in reconstituted systems

  • Circular dichroism to verify proper secondary structure content typical of membrane proteins

For researchers interested in assessing SLC31A2 copper transport function, radioactive 64Cu uptake assays in vesicles or liposomes containing the recombinant protein provide direct evidence of transport activity. Alternatively, indirect measures using copper-sensitive fluorescent probes can assess copper mobilization in cellular compartments.

How can researchers effectively study the interaction between SLC31A2 and cisplatin?

To investigate the relationship between SLC31A2 and cisplatin resistance, researchers should employ multiple complementary approaches:

• Gene Expression Modulation:

  • siRNA or shRNA-mediated knockdown of SLC31A2 in cancer cell lines, followed by cisplatin sensitivity testing

  • Overexpression studies using recombinant SLC31A2 to observe changes in cisplatin uptake and cytotoxicity

  • CRISPR-Cas9 gene editing to create SLC31A2-null cell lines for definitive functional studies

• Cisplatin Uptake and Distribution Analysis:

  • Use ICP-MS (Inductively Coupled Plasma Mass Spectrometry) to quantify total cellular platinum content

  • Subcellular fractionation combined with platinum quantification to determine compartmentalization

  • Fluorescently-labeled cisplatin analogs for real-time imaging of cellular distribution

• Protein Interaction Studies:

  • Co-immunoprecipitation to identify potential binding partners involved in cisplatin trafficking

  • Proximity labeling techniques (BioID or APEX) to map the SLC31A2 interactome in the presence/absence of cisplatin

  • FRET-based approaches to detect direct interactions with cisplatin or other components of transport machinery

The existing finding that SLC31A2 knockdown enhances cisplatin uptake should be validated across multiple cell types, particularly those derived from cancers typically treated with platinum drugs. Time-course studies are essential to distinguish between effects on initial drug uptake versus altered intracellular distribution or efflux.

What approaches are recommended for studying the comparative roles of SLC31A1 (CTR1) and SLC31A2 (CTR2) in copper transport?

To elucidate the distinct and potentially complementary roles of SLC31A1 and SLC31A2 in copper homeostasis, several experimental approaches are recommended:

• Conditional Knockout/Knockdown Studies:

  • Generate cell lines with inducible knockdown of either or both transporters

  • Create double knockout models to assess synthetic effects and reveal compensatory mechanisms

  • Tissue-specific conditional knockouts in animal models to assess organ-specific roles

• Subcellular Localization and Trafficking:

  • Dual-color live cell imaging with differentially tagged SLC31A1 and SLC31A2

  • Immunogold electron microscopy for precise subcellular localization

  • RUSH (Retention Using Selective Hooks) system to track dynamic trafficking of both transporters

• Copper Transport Measurements:

  • Compartment-specific copper sensors to distinguish between cytosolic and vesicular copper pools

  • 64Cu uptake and efflux kinetics in cells with varied expression of each transporter

  • X-ray absorption spectroscopy to determine the oxidation state of copper transported by each protein

• Response to Physiological Stimuli:

  • Compare expression and localization changes under copper deficiency or excess

  • Assess response to hypoxia, oxidative stress, and other conditions that alter copper demands

  • Evaluate interactions with copper chaperones and downstream copper-requiring enzymes

When designing these experiments, it's important to note the fundamental differences in copper binding between these proteins. While studies of prion proteins have shown that copper coordination often involves histidine imidazole groups and deprotonated glycine amides , the precise copper-binding mechanisms in SLC31A2 may differ and should be characterized through site-directed mutagenesis of potential coordinating residues.

The table below summarizes key differences between SLC31A1 and SLC31A2 that should inform experimental design:

How should researchers interpret conflicting data regarding SLC31A2 function in different experimental systems?

When confronted with conflicting data on SLC31A2 function, researchers should consider several factors that might explain discrepancies:

• Cell Type Specificity: SLC31A2 may function differently depending on the cellular context. While ubiquitously expressed, its activity could be modulated by cell-specific factors such as expression levels of copper chaperones, other transporters, or regulatory proteins. Comparing data across multiple cell lines and primary tissues is essential.

• Subcellular Localization Variations: The predominant intracellular localization of SLC31A2 may vary between cell types or experimental conditions. As seen with other transporters, targeting signals can be conditionally masked or exposed, potentially explaining functional differences in various experimental systems.

• Copper Status Dependency: The activity and importance of SLC31A2 may be contingent on cellular copper status. Under normal conditions, its contribution might be minimal, but during copper deprivation or excess, it could assume a more significant role. Experiments should control for and explicitly test various copper conditions.

• Methodological Considerations: Different assays for measuring copper transport have varying sensitivities and limitations. Direct copper measurements using radioactive 64Cu will yield different insights compared to indirect assays using metal-sensitive fluorescent probes or phenotypic readouts of copper-dependent enzymes.

When analyzing apparently contradictory results, a systematic comparison of experimental conditions, particularly the copper concentration, cell type, and detection methods, may reveal conditional aspects of SLC31A2 function that explain the discrepancies.

What are the key considerations when analyzing the relationship between SLC31A2 expression and clinical outcomes in cancer patients?

When analyzing correlations between SLC31A2 expression and cancer outcomes, researchers should address these critical considerations:

• Multivariate Analysis: SLC31A2's prognostic value should be assessed in multivariate models that include established prognostic factors such as stage, grade, and molecular subtypes. This approach determines whether SLC31A2 provides independent prognostic information.

• Expression Contextualization: Both mRNA and protein levels should be evaluated, as post-transcriptional regulation may result in discrepancies. Additionally, consider the subcellular localization of SLC31A2, which may be more informative than total expression levels.

• Cancer Type Specificity: The prognostic significance of SLC31A2 in clear cell renal cell carcinoma may not translate to other cancer types. Copper metabolism and requirements vary across cancer types, necessitating cancer-specific validation.

• Treatment History Confounding: Patient treatment history, particularly exposure to platinum-based chemotherapies, may confound the relationship between SLC31A2 expression and outcomes due to its role in cisplatin resistance.

• Temporal Dynamics: SLC31A2 expression may change during disease progression or in response to treatment. Single time-point measurements might miss critical dynamic changes that influence outcomes.

Researchers should consider using publicly available cancer genomics databases like TCGA and ICGC to perform preliminary analyses across multiple cancer types before investing in prospective studies or detailed molecular characterization of SLC31A2 in specific cancer contexts.

What are the most promising research avenues for elucidating SLC31A2's physiological role?

Several promising research directions could significantly advance our understanding of SLC31A2's physiological functions:

• Conditional and Tissue-Specific Knockout Models: Generating SLC31A2 knockout animal models, particularly with tissue-specific or inducible systems, would overcome potential embryonic lethality issues and reveal tissue-specific functions. This approach has been invaluable for understanding SLC31A1, where whole-body knockouts in mice are lethal at mid-gestation .

• Copper Sensing and Signaling: Investigating SLC31A2's potential role in copper-dependent signaling pathways, particularly in the context of stress responses and cellular adaptation to changing copper availability. Its intracellular localization makes it ideally positioned for such roles.

• Interaction with Copper Chaperones: Characterizing the direct interactions between SLC31A2 and copper chaperones like ATOX1, CCS, and COX17 would illuminate how copper is transferred from intracellular compartments to specific protein targets.

• Vesicular Copper Dynamics: Developing improved methodologies for measuring copper concentrations within specific intracellular compartments (endosomes, lysosomes) would allow direct assessment of SLC31A2's transport activity in its native environment.

• Structural Biology Approaches: Determining the three-dimensional structure of SLC31A2 through cryo-EM or X-ray crystallography would provide critical insights into its transport mechanism and potential for pharmacological modulation. The relatively small size (143 amino acids) makes it a feasible target for structural studies compared to larger transporters.

These approaches, particularly when combined with systems biology techniques that capture global cellular responses to SLC31A2 modulation, could resolve the apparent paradox of a conserved transporter with no obvious impact on cellular copper metabolism.

How might SLC31A2 research contribute to therapeutic approaches for copper-related disorders?

While SLC31A1 (CTR1) has been extensively linked to copper-related disorders like Menkes and Wilson diseases , SLC31A2's potential therapeutic relevance remains largely unexplored but promising:

• Cisplatin Resistance Modulation: The finding that SLC31A2 knockdown enhances cisplatin uptake suggests that targeted inhibition of SLC31A2 could sensitize resistant tumors to platinum-based chemotherapies. Developing small molecule inhibitors specifically targeting SLC31A2 could represent a novel approach to overcome treatment resistance.

• Copper Redistribution in Neurodegeneration: Given the importance of copper dyshomeostasis in neurodegenerative conditions like Alzheimer's and Parkinson's diseases, SLC31A2's role in intracellular copper redistribution could be therapeutically relevant. Compounds that modulate SLC31A2 activity might help normalize copper distribution in affected neurons.

• Biomarker Development: Beyond its prognostic value in renal cell carcinoma , SLC31A2 expression or activity could serve as a biomarker for copper status or drug response in various conditions. Non-invasive methods to assess SLC31A2 status could guide personalized medicine approaches.

• Targeted Copper Delivery: Understanding SLC31A2's transport mechanism could inform the design of copper complexes that selectively enter intracellular compartments, potentially addressing localized copper deficiencies without causing systemic toxicity.

• Copper-Dependent Angiogenesis Modulation: Given SLC31A2's association with angiogenesis through the hypoxia-inducible factor pathway , targeting this transporter could provide a novel approach to modulate pathological angiogenesis in cancer, retinal diseases, and other conditions.

Research in these directions requires deeper characterization of SLC31A2's precise role in copper homeostasis and improved tools for specifically targeting this transporter without affecting other components of copper metabolism.