RCHY1 Human

What is RCHY1 and what are its principal functional characteristics?

RCHY1 is a RING-H2-type protein-ubiquitin E3 ligase that is expressed as multiple isoforms generated through alternative splicing of mRNA transcripts . The protein's primary function involves mediating the ubiquitination of specific target proteins, marking them for degradation by the 26S proteasome. RCHY1 is notably a p53-regulated gene, and multiple studies have demonstrated that it directly binds p53 to promote its ubiquitin-dependent proteasomal degradation, ultimately leading to repression of p53 growth suppressive transcriptional activity . This creates a negative feedback loop in p53 regulation, as RCHY1 itself is regulated by p53. Beyond p53 regulation, RCHY1 also promotes deregulated cell proliferation by targeting the CDK inhibitor, p27 Kip1, for proteasomal degradation . This multifaceted functionality explains why RCHY1 overexpression has been linked to various cancers, including lung and prostate malignancies .

What is the domain architecture of RCHY1 and how does it contribute to function?

RCHY1 comprises three modular domains with distinct structural features that collectively enable its E3 ligase activity. NMR spectroscopy studies have revealed that RCHY1 binds nine zinc ions using various zinc coordination schemes, creating a complex structural scaffold . These domains include:

• N-terminal domain (NTD, residues 1-137)

• RING domain (residues 138-189) - critical for E3 ligase catalytic activity

• C-terminal domain (CTD, residues 137-261) - important for substrate recognition

The protein features a novel left-handed β-spiral structure in which three zinc ions align three consecutive small β-sheets in an interleaved fashion . The RING domain mediates the transfer of ubiquitin from an E2 enzyme to substrate proteins, while the C-terminal zinc-binding module is particularly important for substrate recognition, especially for binding to the tetramerization domain of p53 . This domain organization creates a sophisticated molecular machine capable of recognizing specific substrates and facilitating their ubiquitination.

How does RCHY1 interact with and regulate p53?

The interaction between RCHY1 and p53 is primarily dependent on the C-terminal zinc-binding module of RCHY1, which binds specifically to the tetramerization domain (TET) of p53 . This structural interaction has significant functional implications, as it enables RCHY1 to preferentially ubiquitylate the tetrameric form of p53 both in vitro and in vivo . This selectivity is particularly important because the tetrameric form represents the transcriptionally active configuration of p53 responsible for activating genes involved in cell cycle arrest and apoptosis.

When bound to p53, RCHY1 promotes the addition of ubiquitin molecules to p53, marking it for degradation by the 26S proteasome . This targeted degradation represents a critical control point in p53 regulation. In cancer contexts, RCHY1 overexpression can lead to excessive degradation of p53, compromising its tumor suppressor functions and creating a permissive environment for uncontrolled cell proliferation and accumulation of mutations . The relationship between RCHY1 and p53 is particularly significant in tumors with intact p53, as higher RCHY1 expression levels have been observed to predict poor clinical outcomes in these cases .

What are the optimal antibodies and detection methods for studying RCHY1?

For Western blot analysis, Cell Signaling Technology's RCHY1 Antibody (#5754) has been validated for detecting endogenous levels of total RCHY1 protein . This antibody:

• Is effective at a dilution of 1:1000 for Western blotting

• Can also be used for immunoprecipitation at a 1:50 dilution

• Detects RCHY1 at its expected molecular weight of approximately 29 kDa

• Shows reactivity with human and mouse samples

• Does not detect RCHY1 dimers

When selecting antibodies for RCHY1 detection, researchers should consider specificity, application compatibility, species reactivity, and recognition of specific domains. It is advisable to validate antibody performance in your specific experimental system, possibly using RCHY1 overexpression or knockdown controls to confirm specificity.

What expression systems are available for recombinant RCHY1 protein production?

Recombinant RCHY1 protein has been successfully expressed in E. coli expression systems as demonstrated in multiple studies . For instance, the full-length Pirh2 (RCHY1) and its individual domains have been expressed in E. coli strain BL21 (DE3) with the following parameters:

• Induction with 1 mM IPTG at room temperature for five hours

• Growth in Luria Bertani (LB) media for non-labeled proteins

• For NMR studies, growth in M9 minimal media supplemented with ¹⁵N ammonium chloride (0.8 g/L), ¹³C glucose (2 g/L) and 50 μM ZnSO₄ for uniformly labeled protein

Commercial recombinant human RCHY1 protein preparations are also available, such as the enQuireBio™ Recombinant Human RCHY1 Protein (QP7847-ec-50ug), which is produced by expressing the human RCHY1 DNA sequence in E. coli with a GST tag . The protein has a molecular weight of approximately 25.9 kDa and is typically supplied in a Tris-based buffer containing 50% glycerol .

What are the critical considerations for successful RCHY1 protein purification?

Purifying RCHY1 protein presents several challenges due to its complex domain structure and zinc-binding properties. Key considerations include:

Zinc dependency:

• RCHY1 binds nine zinc ions through various coordination schemes

• Expression and purification buffers should contain appropriate zinc concentrations (10-50 μM ZnCl₂ or ZnSO₄)

• Chelating agents should be avoided in buffers

Purification strategy:

• GST-affinity chromatography has been successfully used for RCHY1 purification

• Thrombin cleavage followed by size exclusion chromatography can effectively remove the GST tag

• A typical purification protocol involves standard GST-affinity chromatography followed by size exclusion using a HiLoad 26/60 Superdex-75 column

Buffer composition:

• Optimal buffer conditions include 50 mM sodium phosphate (pH 7.0), 150 mM KCl, 10 μM ZnCl₂, and 2 mM DTT

• Reducing agents (DTT or β-mercaptoethanol) are important to maintain the integrity of cysteine residues in zinc-binding motifs

• Glycerol (10-50%) improves protein stability during storage

When expressing RCHY1, lower temperatures (room temperature instead of 37°C) typically yield better results for proper protein folding, and the purity of the final product should be at least 80% as determined by SDS-PAGE .

How does RCHY1 dysregulation contribute to cancer development?

RCHY1 dysregulation, particularly overexpression, contributes to cancer development through several interconnected mechanisms:

p53 pathway disruption:

• RCHY1 directly binds p53 and promotes its ubiquitin-dependent proteasomal degradation

• Overexpression of RCHY1 leads to excessive degradation of p53, compromising its tumor suppressor functions including cell cycle arrest, DNA repair, and apoptosis

• This creates a permissive environment for uncontrolled cell proliferation and accumulation of mutations

Cell cycle dysregulation:

• RCHY1 targets the CDK inhibitor p27 Kip1 for proteasomal degradation

• Reduced p27 Kip1 levels promote deregulated cell proliferation by removing inhibition of cyclin-dependent kinases

• This contributes to uncontrolled cell division characteristic of cancer cells

Cancer-specific associations:

• Lung cancer: RCHY1 overexpression has been linked to lung cancer in multiple studies

• Prostate cancer: Evidence suggests RCHY1 overexpression plays a role in prostate cancer progression

• Hepatocellular carcinoma (HCC): RCHY1 function intersects with pathways relevant to HCC development, particularly in p53 regulation

Clinically, higher RCHY1 expression levels predict poor outcomes for cancer patients, especially in those with intact p53 . This suggests that RCHY1 may serve as both a prognostic marker and potential therapeutic target in certain cancer types, especially those relying on p53 inactivation for progression.

What is known about RCHY1's role in viral infections and immune response?

RCHY1 has been identified as a potential biomarker in Ebola virus infection, with its expression patterns potentially serving as predictors of disease outcomes:

Expression signature in Ebola infection:

• RCHY1 was identified among a set of genes with differential expression patterns that distinguished between survivors and non-survivors of Ebola virus infection in anticoagulant-treated non-human primates (NHPs)

• Specifically, RCHY1 showed downregulated expression in survivors compared to non-survivors

• It was part of a minimal gene set of 20 probes that could predict survival outcomes with high accuracy

Predictive biomarker potential:

• RCHY1 was among 10 genes (ACCN1, CEBPE, CRHR2, FAM63A, HMP19, IL2RA, LTF, PSMA1, RCHY1, SLC9A7) with downregulated expression in survivors

• This gene set, along with other genetic markers, correctly classified samples with 100% accuracy in leave-one-out cross-validation

• RCHY1 was also present in a larger predictive signature of 245 unique probes that distinguished among treated survivors, treated non-survivors, and untreated non-survivors

Immune response connection:

• Several genes in the predictive signature, including RCHY1, play roles in immune and/or inflammatory responses

• Non-survivors exhibited different patterns of gene expression related to immune/inflammatory response compared to survivors

• These findings align with the hypothesis that non-survivors experience acute dysregulation of the immune response during Ebola infection

This suggests that RCHY1's role in viral infections may extend beyond its known function in p53 regulation, potentially involving immune system modulation or viral response pathways.

How does RCHY1 fit into the larger network of p53 regulation?

RCHY1 functions within a complex network of E3 ligases that collectively regulate p53 levels and activity:

Multiple E3 ligases targeting p53:

• Mdm2 is the prototypic and primary p53 E3 ligase

• Other E3 ligases including RCHY1 (Pirh2), COP1, TOPORS, and ARF-BP1 also target p53

• This multi-ligase system provides redundancy and allows for context-specific regulation

RCHY1's specific role:

• RCHY1 preferentially ubiquitylates the tetrameric (transcriptionally active) form of p53

• This specificity suggests RCHY1 has a particular role in regulating active p53

• RCHY1 is itself a p53-regulated gene, creating a negative feedback loop

Functional significance:

• The C-terminal zinc-binding module of RCHY1 specifically binds to the tetramerization domain of p53

• This interaction enables RCHY1 to regulate the protein turnover of the transcriptionally active form of p53 in cells

• In cancer contexts, constitutive overexpression of RCHY1 can disrupt this feedback mechanism, maintaining persistently low p53 levels

Understanding this network has significant implications for cancer biology and therapeutics. Since higher RCHY1 expression levels predict poor clinical outcomes for cancer patients with intact p53 , targeting RCHY1 could potentially restore p53 function in these tumors, offering a complementary approach to existing p53-focused therapeutic strategies.

What are the structural mechanisms underlying RCHY1's substrate specificity?

The structural basis for RCHY1's substrate specificity involves several sophisticated mechanisms:

Domain-specific interactions:

• The C-terminal zinc-binding module of RCHY1 specifically interacts with the tetramerization domain (TET) of p53

• This domain selectivity explains why RCHY1 preferentially ubiquitylates the tetrameric form of p53

• The tetrameric form is the transcriptionally active configuration of p53, suggesting RCHY1 specifically regulates active p53

Zinc-dependent structural features:

• RCHY1 binds nine zinc ions using various zinc coordination schemes

• This complex zinc-binding pattern creates a unique structural scaffold

• The protein features a novel left-handed β-spiral in which three zinc ions align three consecutive small β-sheets in an interleaved fashion

• These precise arrangements of zinc-binding motifs likely create specific recognition surfaces for different substrates

Modular domain architecture:

• RCHY1 comprises three distinct domains with specialized functions:

  • N-terminal domain (NTD, residues 1-137)

  • RING domain (residues 138-189) - mediates E2 binding and catalysis

  • C-terminal domain (CTD, residues 137-261) - critical for substrate recognition

This complex structural organization allows RCHY1 to recognize specific substrates with high precision while maintaining its catalytic E3 ligase activity. Understanding these structural mechanisms could inform the design of specific inhibitors that disrupt recognition of particular substrates while preserving others, potentially allowing for more targeted therapeutic approaches.

What is the relationship between RCHY1 and other biomarkers in disease prognostication?

RCHY1 shows potential as a prognostic biomarker in several disease contexts, particularly when considered alongside other molecular markers:

Cancer prognosis:

• Higher RCHY1 expression levels predict poor clinical outcomes for cancer patients, especially in those with intact p53

• This relationship suggests that combining RCHY1 expression data with p53 mutational status could provide more refined prognostication

• In hepatocellular carcinoma, RCHY1 function intersects with pathways involving RRS1, which promotes HCC development through attenuating the RPL11-MDM2-p53 pathway

Viral infection outcomes:

• In Ebola virus infection, RCHY1 was part of a minimal gene set of 20 probes that could predict survival outcomes with high accuracy

• This gene set included other markers such as CLDN3, ILF2, ILF3, NDUFA12, RUVBL2, SLC38A5 (upregulated in survivors) and ACCN1, CEBPE, CRHR2, FAM63A, HMP19, IL2RA, LTF, PSMA1, SLC9A7 (downregulated in survivors)

• Hierarchical clustering and leave-one-out cross-validation confirmed that this minimal gene set correctly classified samples with 100% accuracy

Common transcriptional regulators:

• Research has identified transcriptional regulators with downstream targets whose expression correlated with survival in Ebola infection, including CCAAT/enhancer-binding protein alpha (CEBPA), tumor protein 53 (p53), megakaryoblastic leukemia 1 (MKL1) and myocardin-like protein 2 (MKL2)

• These pathways could reveal transcriptional signatures specific to infection and host immune response that involve RCHY1

The integration of RCHY1 status with other biomarkers could significantly enhance prognostic accuracy, enabling more personalized treatment approaches in both cancer and infectious disease contexts.

How do zinc-binding properties influence RCHY1's structural integrity and function?

Zinc coordination plays a fundamental role in RCHY1's structure and catalytic function:

Complex zinc coordination pattern:

• RCHY1 binds nine zinc ions using various zinc coordination schemes

• This includes a RING domain and a novel left-handed β-spiral structure

• In the β-spiral, three zinc ions align three consecutive small β-sheets in an interleaved fashion

• This complex zinc-binding pattern creates the structural scaffold necessary for RCHY1's function

Critical role in RING domain function:

• The RING domain of RCHY1 contains zinc-coordinating motifs essential for E3 ligase activity

• Zinc coordination in RING domains typically creates a platform for E2 enzyme binding

• Proper zinc coordination is required for the transfer of ubiquitin from the E2 to the substrate

• The RING domain (residues 138-189) is specifically implicated in the catalytic process of ubiquitin transfer

Substrate recognition dependence:

• The C-terminal zinc-binding module is critical for substrate recognition, particularly for binding to the tetramerization domain of p53

• Zinc coordination likely creates specific structural features that mediate protein-protein interactions

• Alterations in zinc coordination could affect substrate specificity or binding affinity

For researchers working with RCHY1, maintaining appropriate zinc concentrations in experimental buffers is crucial. Typical buffers include 10-50 μM ZnCl₂ to ensure proper protein folding and function . Additionally, reducing agents (e.g., DTT) are important to maintain the integrity of zinc-coordinating cysteine residues, and zinc chelators should be avoided in experimental conditions.

How might targeting RCHY1 affect p53-dependent cancer therapies?

Targeting RCHY1 could significantly impact p53-dependent cancer therapies through several mechanisms:

Enhancement of p53 activity:

• Inhibiting RCHY1 would reduce p53 ubiquitination and degradation

• This would lead to increased p53 levels and enhanced p53-dependent responses

• This approach would be particularly relevant for tumors with wild-type p53 and RCHY1 overexpression

• Clinical correlations show that higher RCHY1 expression levels predict poor clinical outcomes for patients with cancer, especially in those with intact p53

Complementary approach to existing therapies:

• RCHY1 inhibition could complement MDM2 inhibitors currently in development

• While MDM2 is the primary regulator of p53, RCHY1 provides an additional layer of p53 regulation

• Combined inhibition of MDM2 and RCHY1 might achieve more robust p53 stabilization

• This could help address resistance mechanisms to MDM2 inhibitors

Context-dependent considerations:

• Effectiveness would depend on p53 status (wild-type vs. mutant)

• Most beneficial in cancers with wild-type p53 and RCHY1 overexpression

• May be particularly relevant for lung and prostate cancers with documented RCHY1 overexpression

• In hepatocellular carcinoma, targeting RCHY1 could potentially disrupt the pathways attenuating the RPL11-MDM2-p53 pathway

The therapeutic potential of targeting RCHY1 is supported by findings that RCHY1 preferentially ubiquitylates the tetrameric form of p53, suggesting it regulates the transcriptionally active form of p53 in cells . This specificity could allow for more precise modulation of p53 function in therapeutic contexts.

What potential exists for RCHY1 as a diagnostic or prognostic marker?

RCHY1 holds considerable potential as both a diagnostic and prognostic marker in several disease contexts:

Cancer prognosis:

• Higher RCHY1 expression levels predict poor clinical outcomes for cancer patients, especially those with intact p53

• This correlation could help identify high-risk patients who might benefit from more aggressive therapy

• Particularly relevant in lung and prostate cancers where RCHY1 overexpression has been documented

Treatment stratification:

• RCHY1 expression might help predict response to therapies that rely on p53 function

• Tumors with high RCHY1 and wild-type p53 might respond differently to p53-activating therapies

• Could identify patients likely to benefit from therapeutic strategies targeting the RCHY1-p53 axis

Infectious disease applications:

• RCHY1 was identified in gene expression signatures predicting survival in Ebola virus infection

• It was part of a minimal gene set that correctly classified survival outcomes with 100% accuracy

• Could contribute to prognostic models for viral infection outcomes and potentially guide treatment decisions

Implementation considerations:

• Detection methods: RCHY1 can be detected via immunohistochemistry, RT-PCR, or RNA-seq for expression analysis

• The Cell Signaling Technology RCHY1 Antibody (#5754) has been validated for detecting endogenous levels of total RCHY1 protein in western blot and immunoprecipitation applications

• Clinical validation: Large, prospective studies would be needed to confirm prognostic/predictive value

Integration with other markers:

• Combining RCHY1 status with p53 mutation status could provide more refined prognostication

• Incorporating RCHY1 into multi-gene signatures might improve predictive accuracy

• The minimal gene set identified in Ebola research demonstrates the power of such integrated approaches

The potential of RCHY1 as a biomarker warrants further investigation in large, prospective clinical studies to validate its utility and establish standardized assessment methods across different disease contexts.