Recombinant Human Leucine-rich repeat-containing protein 16C (RLTPR), partial

What are the key structural domains of human RLTPR protein?

Human RLTPR consists of four major functional domains: an RGD domain, a leucine-rich repeat (LRR) domain, a tropomodulin domain, and a proline-rich region. The LRR domain is particularly important, forming a characteristic horseshoe-shaped structure that facilitates protein-protein interactions. Each of these domains contributes to RLTPR's scaffolding function, with the noncanonical pleckstrin-homology domain, LRR domain, and proline-rich region being mandatory for CD28 co-stimulation in T cells .

How does RLTPR function in T cell signaling pathways?

RLTPR acts primarily as a scaffolding protein that bridges CD28 to the CARD11/CARMA1 cytosolic adaptor and the NF-κB signaling pathway. In this capacity, it facilitates signal transduction following T cell receptor engagement and CD28 co-stimulation. Though RLTPR was initially thought to function as an actin-uncapping protein, research has demonstrated that this property is dispensable for CD28 co-stimulation in both mouse and human T cells, suggesting that its scaffolding role predominates during T cell activation .

What approaches are recommended for studying RLTPR protein-protein interactions?

When investigating RLTPR interactions, affinity purification followed by mass spectrometry analysis has proven effective. This approach revealed RLTPR's association with CARD11/CARMA1 and identified novel proteins within the CD28 signaling pathway. For functional validation, co-immunoprecipitation experiments can confirm direct interactions, while CRISPR-Cas9-mediated knockout models allow assessment of interaction dependencies. Researchers should control for potential scaffold-mediated indirect interactions by including appropriate negative controls and considering cross-linking strategies to capture transient associations .

How should researchers design experiments to evaluate RLTPR's role in T cell function?

A comprehensive experimental design should include:

• Functional assays measuring T cell activation (CD69, CD25 expression)

• Proliferation assays with anti-CD3/CD28 stimulation

• Cytokine production analysis (particularly IL-2, IFN-γ)

• Intracellular signaling assessment (NF-κB activation, PKCθ translocation)

• T cell subset differentiation (Th1, Th17, Treg development)

Both gain-of-function (overexpression) and loss-of-function (CRISPR knockout, siRNA) approaches should be employed. Critical controls include isolated CD3 stimulation without CD28 co-stimulation and comparison with known CD28 pathway inhibitors .

What are the most effective methods for producing recombinant RLTPR for structural studies?

For structural studies of recombinant RLTPR, a bacterial expression system using E. coli BL21(DE3) with domain-specific constructs is recommended, as the full-length protein (1435 amino acids) may present solubility challenges. Domain-specific expression (particularly of the LRR domain) in mammalian HEK293T cells with an N-terminal His-tag and C-terminal FLAG-tag allows for tandem purification. Protein stability is enhanced by inclusion of 10% glycerol and 1mM DTT in all buffers. For structural determination, a combination of X-ray crystallography for individual domains and cryo-EM for larger assemblies has proven most informative for LRR-containing proteins .

How does RLTPR contribute to CD28 co-stimulation at the molecular level?

RLTPR functions as a molecular bridge, directly linking CD28 to CARD11/CARMA1 adaptor and the NF-κB signaling pathway. Following CD28 engagement, RLTPR facilitates the recruitment of signaling molecules that lead to PKCθ activation and subsequent NF-κB signaling. The LRR domain of RLTPR is crucial for this function, as it mediates specific protein-protein interactions within the signaling complex. Mouse studies have suggested that Rltpr may also control CD28 internalization, potentially regulating signal duration and intensity. The scaffolding function of RLTPR appears to be its primary role in co-stimulation, as its actin-uncapping properties are dispensable for this process .

What other proteins interact with RLTPR in the immune signaling network?

Beyond the established interactions with CD28 and CARD11/CARMA1, mass spectrometry analysis has identified several previously unknown RLTPR interactors:

These interactions suggest RLTPR is part of a complex signaling network extending beyond the canonical CD28 pathway .

What approaches should be used to functionally characterize RLTPR mutations?

To functionally characterize RLTPR mutations, a comprehensive approach involving both in silico and experimental methods is recommended:

• Computational structure prediction: Use homology modeling (e.g., Phyre2 Protein Fold Recognition Server) to assess potential structural impacts, particularly for mutations in the LRR domain where the conserved leucine residues are crucial for maintaining the hydrophobic core.

• Expression analysis: Compare expression levels of wild-type and mutant RLTPR through Western blotting and RT-qPCR to determine if mutations affect protein stability or expression.

• Protein-protein interaction assays: Use co-immunoprecipitation and proximity ligation assays to assess whether mutations disrupt RLTPR's interactions with CD28, CARD11/CARMA1, or other partners.

• Functional T cell assays: Compare CD28-dependent functions in T cells expressing wild-type versus mutant RLTPR, including:

  • NF-κB activation

  • Cytokine production

  • Proliferation responses

  • Regulatory T cell development

• In vivo models: Generate knock-in mice expressing mutations of interest to assess physiological impact .

How do variants in the LRR domain specifically affect RLTPR function?

Variants in the LRR domain can significantly impact RLTPR function due to the domain's critical role in protein-protein interactions. The LRR domain has a canonical sequence motif (LxxLxLxxN/CxL) where conserved leucines contribute to the hydrophobic core, providing structural stability. Mutations affecting these conserved leucines (such as the p.Leu639His variant identified in Norwegian patients) can destabilize the three-dimensional horseshoe-shaped structure of the LRR domain.

Mouse studies with the p.L432P variant (affecting a conserved leucine in LRR 8) have demonstrated that such mutations can disrupt Rltpr's ability to link CD28 to PKCθ and Carma1. The functional consequences include:

• Impaired regulatory T cell development

• Reduced CD4+ memory T cell populations

• Deficient IFN-γ synthesis in CD4+ T cells and NK cells

• Compromised antiviral and antifungal immunity

These effects are consistent with the clinical phenotypes observed in patients with RLTPR deficiency, including susceptibility to viral infections (warts, molluscum contagiosum) and dermatitis .

What are the immunological phenotypes associated with RLTPR deficiency?

RLTPR deficiency produces a distinct immunological profile that includes:

Clinically, patients present with warts, molluscum contagiosum, and dermatitis from early childhood, reflecting impaired viral immunity. This represents the first described primary immunodeficiency affecting the human CD28 pathway in T cells .

How can researchers differentiate RLTPR-related immunodeficiency from other similar conditions?

RLTPR deficiency presents as a distinct clinical entity that must be differentiated from other immunodeficiencies with similar presentations. Key diagnostic approaches include:

• Genetic analysis: Next-generation sequencing, particularly whole exome sequencing, to identify RLTPR/CARMIL2 variants.

• Immunophenotyping: Flow cytometry analysis focusing on regulatory T cells, memory CD4+ T cells, and follicular T cells, which are characteristically reduced in RLTPR deficiency.

• Functional assays: Assessment of T cell proliferation and cytokine production in response to CD3/CD28 stimulation.

• Differential molecular diagnosis: Exclusion of variants in genes associated with similar phenotypes:

  • CXCR4

  • DOCK8

  • GATA2

  • MAGT1

  • MCM4

  • STK4

  • RHOH

  • TMC6

  • TMC8

The presentation of warts, molluscum contagiosum, dermatitis, and specific T cell subset abnormalities, combined with genomic evidence of CARMIL2/RLTPR variants, provides a definitive diagnosis .

What strategies can address challenges in producing functional recombinant RLTPR protein?

Production of functional recombinant RLTPR presents several challenges due to its size (1435 amino acids) and multiple domains. Effective troubleshooting strategies include:

• Domain-specific expression: Rather than attempting full-length protein expression, focus on individual domains (particularly the LRR domain) which tend to be more stable and soluble.

• Expression system optimization:

  • For structural studies: Insect cell systems (Sf9, High Five) typically yield higher quantities of properly folded protein than bacterial systems

  • For functional studies: Mammalian expression systems (HEK293T, CHO) ensure proper post-translational modifications

• Solubility enhancement:

  • Add solubility tags (MBP, SUMO, thioredoxin) at the N-terminus

  • Include 10% glycerol and reducing agents in purification buffers

  • Optimize purification at 4°C to minimize degradation

  • Consider refolding strategies for inclusion body-derived protein

• Activity confirmation: Verify functional activity through specific protein-protein interaction assays with known binding partners (CD28, CARD11) .

How can researchers assess RLTPR function in T cell subsets with limited cell numbers?

When working with limited primary T cell numbers, particularly from rare patient samples or specialized T cell subsets, modified approaches are necessary:

• Microfluidic-based single-cell analysis: Platforms like Fluidigm allow assessment of signaling pathway activation in minimal cell numbers.

• Phospho-flow cytometry: Multiplexed phospho-protein detection enables simultaneous assessment of multiple signaling nodes downstream of RLTPR with as few as 1×10^4 cells per condition.

• Miniaturized functional assays:

  • Microscale proliferation assays in 384-well formats

  • Cytokine secretion using highly sensitive single-molecule arrays (Simoa)

  • CRISPR-mediated gene editing in primary T cells using ribonucleoprotein delivery for improved efficiency in limited cell numbers

• Ex vivo expansion: When appropriate, limited expansion of primary T cells with IL-2 and artificial APCs before functional assessment can increase cell numbers without significantly altering the phenotype of interest .

What emerging technologies might advance our understanding of RLTPR function?

Several cutting-edge technologies show promise for deepening our understanding of RLTPR biology:

• Cryo-electron microscopy (cryo-EM): For resolving the full-length RLTPR structure and its complexes with binding partners, particularly in different activation states.

• Proximity labeling proteomics (BioID, APEX): To identify transient or context-specific RLTPR interactors in living cells under various stimulation conditions.

• Single-molecule imaging: To visualize RLTPR dynamics during T cell activation in real-time, potentially revealing spatial and temporal regulation of signaling complexes.

• CRISPR-based screening: Domain-focused mutagenesis screens to map functionally critical residues beyond the known leucine-rich repeats.

• Patient-derived induced pluripotent stem cells (iPSCs): For generating T cell models carrying patient-specific RLTPR variants to study developmental and functional consequences in a controlled genetic background.

• Mathematical modeling of signaling networks: To predict how RLTPR scaffold properties influence signal integration, amplification, and duration in the CD28 pathway .

What unresolved questions remain in RLTPR research?

Despite significant advances, several key questions about RLTPR function remain to be addressed:

• Cell type-specific functions: How does RLTPR function in B cells, which express the protein but not CD28? What are its CD28-independent roles?

• Regulatory mechanisms: How is RLTPR expression and function regulated at transcriptional, post-transcriptional, and post-translational levels?

• Structural dynamics: How does RLTPR conformation change upon T cell activation, and how do these changes regulate its scaffolding properties?

• Evolutionary conservation: Given the differences between human and mouse RLTPR functions in B cells, what are the species-specific adaptations of this protein?

• Therapeutic targeting: Can RLTPR-dependent pathways be modulated for therapeutic benefit in immunodeficiency or autoimmune conditions?

• Signaling kinetics: How does RLTPR scaffolding influence the temporal dynamics of CD28 co-stimulation and downstream effector functions?