Recombinant Human Leucine-rich repeat-containing protein 55 (LRRC55)

What is LRRC55 and what is its structural organization?

LRRC55 (Leucine-rich repeat-containing protein 55) is a transmembrane protein of approximately 35 kDa belonging to a family of paralogous proteins including LRRC26, LRRC38, and LRRC52, which share 30-40% amino acid sequence similarity. The protein contains a predicted extracellular leucine-rich repeat (LRR) domain with a single transmembrane topology .

The LRR domain in LRRC55 comprises six LRR units in the middle flanked by two cysteine-rich regions called LRRNT and LRRCT on the N- and C-terminal sides, respectively. The LRR units contain the consensus sequence LxxLxLxxN (where x can be any amino acid) . This structural organization is common among LRR-containing proteins and is critical for protein-protein interactions.

Where is LRRC55 expressed in human tissues?

LRRC55 exhibits a highly tissue-specific expression pattern. While its family members show distinct tissue distributions (LRRC26 and LRRC38 mainly in secretory glands, LRRC52 predominantly in testis), LRRC55 is primarily expressed in brain tissue .

Beyond its neuronal expression, significant upregulation of LRRC55 occurs in pancreatic islets during specific physiological states. For instance, during pregnancy, LRRC55 expression increases by more than 60-fold in pancreatic islets in a prolactin receptor (PrlR)-dependent manner . This dramatic and tissue-specific upregulation suggests important functional roles in both neuronal tissues and pancreatic islets.

How does LRRC55 relate to other leucine-rich repeat proteins?

LRRC55 is part of a family of LRR-containing proteins that function as regulatory subunits of BK potassium channels. Among approximately 400 LRR-containing proteins in the human protein database, LRRC55 belongs to a specific subgroup that includes LRRC26, LRRC38, and LRRC52 . These proteins share several key characteristics:

• Similar protein size of approximately 35 kDa

• Predicted extracellular LRR domain structure

• Single transmembrane topology

• 30-40% amino acid sequence similarity

• Function as BK channel γ-subunits

The amino acid sequences of these proteins show high similarity in the structurally determinant residues of their LRR domains but become more divergent in the non-LRR regions . This selective conservation suggests evolutionary pressure to maintain certain structural features while allowing functional specialization.

How can recombinant LRRC55 be expressed in laboratory settings?

Recombinant expression of LRRC55 can be achieved through several methodological approaches based on techniques used for similar proteins:

• Cloning and Vector Construction:

  • Human LRRC55 cDNA can be subcloned into mammalian expression vectors such as pCDNA6

  • C-terminal tags (FLAG, V5) may be attached to facilitate detection and purification

  • For co-expression studies, fusion constructs encoding precursor fusion proteins of target proteins and LRRC55 can facilitate assembly

• Cell Culture Expression Systems:

  • HEK-293 cells can be transfected with LRRC55-containing plasmids using lipofection reagents like Lipofectamine 2000

  • Transfected cells can be used for electrophysiological assays within 16-72 hours or biochemical assays at approximately 48 hours post-transfection

  • Adenoviral vectors have been successfully used to overexpress LRRC55 in INS-1-832/13 cells and mouse islets

• Purification Approaches:

  • Epitope tags facilitate affinity purification

  • Appropriate detergents must be selected to maintain protein stability during membrane protein extraction

The choice of expression system should be guided by the specific experimental endpoints, whether structural studies, functional assays, or protein-protein interaction analyses.

What techniques are used to quantify LRRC55 expression?

Accurate quantification of LRRC55 expression requires specific methodologies for both mRNA and protein detection:

• Quantitative Real-Time PCR (qRT-PCR):

  • First-strand cDNA synthesis from total RNA using reverse transcriptase with oligo(dT) primers

  • TaqMan real-time PCR with specific primers and probes designed to span exon junctions (LRRC55 is encoded by two exons)

  • Example primer design strategy based on related proteins:

    • Forward primer targeting exon-junction region

    • Reverse primer in second exon

    • Probe spanning the exon-exon junction for specificity

  • Normalization with endogenous controls such as human RPLPO (large ribosomal protein)

• Protein Detection Methods:

  • Western blotting with antibodies against LRRC55 or epitope tags

  • Immunohistochemistry for tissue localization studies

  • Flow cytometry for quantifying expression in cell populations

A comprehensive quantification table from a study examining expression across tissues would include:

What functional assays are appropriate for studying LRRC55 activity?

Several functional assays can effectively evaluate LRRC55 activity and its cellular effects:

• Apoptosis and Cell Survival Assays:

  • TUNEL staining to detect DNA fragmentation in apoptotic cells

  • Caspase-3 activation measurement using fluorogenic substrates

  • Cell viability assays under stress conditions (e.g., palmitate or thapsigargin treatment)

• ER Stress Pathway Analysis:

  • Western blotting for ER stress markers (IRE-1α, CHOP, p-eIF2/eIF2)

  • Analysis of Bax/Bcl-2 ratio as indicator of apoptotic signaling

  • PCR-based detection of XBP1 splicing

• Calcium Homeostasis Measurement:

  • Fluorescent calcium indicators to monitor intracellular calcium dynamics

  • Calcium depletion assays under glucolipotoxicity conditions

• Electrophysiological Studies:

  • Patch-clamp recordings to assess BK channel function when co-expressed with LRRC55

  • Analysis of channel activation kinetics and voltage dependence

• Stress Response Models:

  • Palmitate treatment (0.5 mM, 24-72 hours) to induce glucolipotoxicity

  • Thapsigargin exposure (100-300 nM) to trigger ER stress

These methodologies provide complementary insights into LRRC55 function, from molecular interactions to cellular protective effects.

What is the role of LRRC55 in BK channel regulation?

LRRC55 functions as a γ-subunit of BK (Big Potassium) channels, though the specific mechanisms of its regulatory effects require further characterization. Based on its structural similarity to other family members, LRRC55 likely modulates BK channel gating properties .

The leucine-rich repeat domain in LRRC55 shares structural features with its paralogous proteins (LRRC26, LRRC38, and LRRC52), which are established BK channel γ-subunits . These γ-subunits significantly influence the voltage and calcium dependence of BK channel activation, typically shifting the activation voltage to more hyperpolarized potentials.

Given LRRC55's predominant expression in brain tissue, it likely plays a tissue-specific role in modulating neuronal BK channels, potentially affecting:

• Neuronal excitability

• Action potential repolarization

• Calcium signaling

• Neurotransmitter release

The specific electrophysiological properties conferred by LRRC55 association with BK channels may differ from those of other family members, reflecting its specialized function in neuronal tissues.

How does LRRC55 protect pancreatic β-cells from apoptosis?

LRRC55 demonstrates significant anti-apoptotic properties in pancreatic β-cells through multiple mechanisms:

• Modulation of ER Stress Pathways:

  • Overexpression of LRRC55 attenuates the expression of key ER stress markers including IRE-1α and CHOP

  • LRRC55 affects the p-eIF2/eIF2 ratio, influencing the PERK branch of the unfolded protein response

  • These effects prevent the transition from adaptive UPR to unresolved ER stress that triggers apoptosis

• Regulation of Apoptotic Mediators:

  • LRRC55 overexpression leads to decreased activation of caspase-9, a key initiator caspase

  • Reduces Bax/Bcl-2 ratio, shifting the balance toward anti-apoptotic signaling

  • Decreases activation of caspase-3, the primary executioner caspase

• Calcium Homeostasis Preservation:

  • LRRC55 attenuates calcium depletion induced by glucolipotoxicity

  • Maintaining proper calcium levels is crucial for ER function and preventing ER stress-induced apoptosis

• Protection Against Multiple Stressors:

  • LRRC55 provides protection against apoptosis induced by palmitate (modeling glucolipotoxicity)

  • Also protects against thapsigargin-induced apoptosis (a SERCA inhibitor that induces ER stress)

These protective mechanisms are particularly relevant during pregnancy, when pancreatic β-cells face increased demand for insulin production, potentially explaining the dramatic upregulation of LRRC55 in islets during this physiological state.

What role does LRRC55 play during pregnancy and metabolic stress?

LRRC55 appears to play a crucial role in pancreatic islet adaptation during pregnancy and under metabolic stress conditions:

• Pregnancy-Related Upregulation:

  • LRRC55 expression increases by >60-fold in pancreatic islets during pregnancy (day 15)

  • This upregulation is prolactin receptor (PrlR)-dependent, as demonstrated by reduced expression in PrlR+/- mice

  • The dramatic increase is specific to islets and not observed in other tissues

• Adaptive Response to β-Cell Stress:

  • During pregnancy, β-cells face increased insulin production demands

  • This increased protein synthesis burden triggers unfolded protein response (UPR)

  • Markers of UPR (IRE-1α, CHOP) increase by pregnancy day 12

  • LRRC55 appears to prevent progression from UPR to apoptosis

• Response to Metabolic Disease:

  • LRRC55 is upregulated by >10-fold in islets from obese db/db mice, a model of metabolic syndrome and diabetes

  • Expression increases within 24 hours of palmitate exposure in mouse islets

  • Similar upregulation occurs following thapsigargin treatment, suggesting LRRC55 induction is part of the cellular stress response

This expression pattern suggests LRRC55 functions as part of an adaptive response mechanism that protects β-cells during periods of increased metabolic demand or stress. The dramatic upregulation during pregnancy correlates with the known resistance of pregnant mice to β-cell apoptosis, supporting LRRC55's role as a prosurvival factor.

What is the relationship between LRRC55 and endoplasmic reticulum stress?

LRRC55 has a complex bidirectional relationship with endoplasmic reticulum (ER) stress:

• LRRC55 Induction by ER Stress:

  • LRRC55 expression increases significantly following treatment with thapsigargin, a SERCA inhibitor that triggers ER stress

  • Palmitate exposure, which causes ER stress in β-cells, also increases LRRC55 expression

  • This suggests LRRC55 is part of the cellular response to ER stress

• LRRC55 Modulation of UPR Components:

  • When overexpressed, LRRC55 attenuates expression of key ER stress pathway components:

    • Reduced IRE-1α expression, affecting the IRE1α-XBP1 branch of UPR

    • Decreased CHOP expression, a pro-apoptotic transcription factor induced during unresolved ER stress

    • Trend toward lower p-eIF2/eIF2 ratio, suggesting effects on PERK-eIF2α signaling

• Prevention of ER Stress-Induced Apoptosis:

  • LRRC55 overexpression protects against thapsigargin-induced apoptosis

  • It prevents the transition from adaptive UPR to unresolved ER stress leading to cell death

  • This protective effect involves reduction in pro-apoptotic signals downstream of ER stress

• Calcium Homeostasis Effects:

  • LRRC55 attenuates calcium depletion induced by glucolipotoxicity

  • ER calcium depletion is a key trigger for ER stress

  • This suggests LRRC55 may prevent ER stress initiation by maintaining calcium homeostasis

The dual nature of this relationship (LRRC55 being both induced by ER stress and acting to attenuate it) suggests LRRC55 functions as part of a negative feedback regulatory mechanism to modulate ER stress responses and protect cells from excessive ER stress-induced damage.

What structural insights guide our understanding of LRRC55 function?

While detailed structural information specific to LRRC55 is limited in the provided research, several structural insights can be inferred:

• LRR Domain Organization:

  • LRRC55 contains a leucine-rich repeat domain comprising six LRR units

  • These LRR units are flanked by cysteine-rich LRRNT and LRRCT regions on the N- and C-terminal sides

  • The LRR units contain a consensus sequence of LxxLxLxxN (where x can be any amino acid)

• Structural Modeling Approaches:

  • Homology modeling approaches similar to those used for LRRC26 could be employed

  • Potential templates include crystal structures of hagfish variable lymphocyte receptor B (for LRRNT and LRR units) and mouse TLR4 (for LRRCT region)

  • Tools like SWISS MODEL and SWISS-pdb viewer can facilitate model development

• Transmembrane Topology:

  • LRRC55 has a predicted single transmembrane domain

  • The LRR domain is likely extracellular, similar to other family members

  • This topology positions LRRC55 to interact with the extracellular domains of BK channels

• Structural Basis for Functional Effects:

  • The LRR domain likely mediates protein-protein interactions

  • Structural studies of related proteins suggest the curved LRR domain creates an ideal interaction surface

  • The specific arrangement of leucine-rich repeats may determine binding specificity and affinity

More detailed structural characterization through techniques like X-ray crystallography or cryo-electron microscopy would significantly advance our understanding of LRRC55's molecular mechanisms of action.

How does LRRC55 expression change in pathological conditions?

LRRC55 expression demonstrates significant alterations in response to various pathological conditions:

• Diabetes and Metabolic Syndrome:

  • LRRC55 is upregulated by >10-fold in islets of obese db/db mice, a model of metabolic syndrome and diabetes

  • This suggests LRRC55 induction as part of the cellular response to metabolic stress

• Cellular Stress Conditions:

  • Exposure to palmitate (0.5 mM), mimicking lipotoxicity found in diabetes, increases LRRC55 expression in islets within 24 hours

  • Treatment with thapsigargin, which induces ER stress similar to that observed in various disease states, also increases LRRC55 expression

  • These responses indicate LRRC55 upregulation as part of a broader cellular stress response

• Protective Response Analysis:

  • The anti-apoptotic properties of LRRC55 suggest its upregulation represents a compensatory protective mechanism

  • This upregulation may be insufficient to prevent disease progression in chronic conditions

  • The temporal dynamics of LRRC55 expression throughout disease progression remain to be fully characterized

• Potential Therapeutic Implications:

  • The protective effects of LRRC55 against ER stress and apoptosis suggest it may be a therapeutic target

  • Further augmentation of LRRC55 expression or function could potentially enhance β-cell survival in diabetes

  • The search results specifically mention LRRC55 as "a potential therapeutic target in diabetes by reducing ER stress and promoting β-cell survival"

These expression changes highlight LRRC55's potential role as a stress-responsive factor whose upregulation may represent an adaptive response to cellular stress in pathological conditions.

What methodological advances would enhance LRRC55 research?

Several advanced methodological approaches could significantly advance LRRC55 research:

• Structural Biology Techniques:

  • Cryo-electron microscopy for determining LRRC55 structure alone or in complex with BK channels

  • X-ray crystallography of isolated LRRC55 domains

  • Hydrogen-deuterium exchange mass spectrometry to map protein interactions

  • Molecular dynamics simulations to predict conformational changes and interaction interfaces

• CRISPR-Based Genome Engineering:

  • Generation of LRRC55 knockout cell lines and animal models

  • Creation of knock-in models with fluorescent or epitope tags

  • Introduction of specific mutations to study structure-function relationships

  • Base editing for precise modification of LRRC55 regulatory elements

• Single-Cell Analysis Technologies:

  • Single-cell RNA sequencing to examine LRRC55 expression heterogeneity

  • Single-cell proteomics to analyze LRRC55 protein levels and modifications

  • Spatial transcriptomics to map LRRC55 expression patterns within complex tissues

• Advanced Functional Imaging:

  • Super-resolution microscopy for nanoscale visualization of LRRC55 localization

  • FRET/BRET analysis to study LRRC55 protein interactions in live cells

  • Calcium imaging combined with LRRC55 manipulation to assess effects on calcium dynamics

• Comprehensive Omics Approaches:

  • Proteomics studies to identify LRRC55 interaction partners

  • Phosphoproteomics to map LRRC55 phosphorylation sites and signaling networks

  • Transcriptomics following LRRC55 manipulation to identify downstream effectors

These methodological advances would provide deeper insights into LRRC55 biology, from molecular structure to physiological function, helping to bridge current knowledge gaps.

What are the primary research gaps in LRRC55 biology?

Despite growing understanding of LRRC55, several significant knowledge gaps remain:

• Molecular Mechanisms:

  • The precise mechanism by which LRRC55 modulates BK channel function remains incompletely characterized

  • How LRRC55 interfaces with ER stress pathways at the molecular level is unclear

  • The signaling mechanisms downstream of LRRC55 require further elucidation

• Physiological Roles:

  • While LRRC55 is highly expressed in brain tissue, its neuronal functions remain largely unexplored

  • The role of LRRC55 in tissues beyond pancreatic islets and brain requires investigation

  • Whether LRRC55 plays roles in development, aging, or tissue homeostasis is unknown

• Tissue-Specific Functions:

  • The functional significance of LRRC55's predominant expression in brain tissue needs clarification

  • How LRRC55 function differs between neurons and pancreatic β-cells is not well understood

  • Whether LRRC55 has undiscovered roles in other tissues with lower expression levels

• Regulation of LRRC55:

  • While prolactin receptor signaling regulates LRRC55 in pregnancy, other transcriptional regulatory mechanisms are largely unknown

  • Post-translational modifications that might regulate LRRC55 function have not been characterized

  • The half-life and degradation pathways of LRRC55 protein remain to be determined

• Clinical Relevance:

  • The potential role of LRRC55 dysfunction in human diseases has not been established

  • Whether LRRC55 genetic variants contribute to disease susceptibility needs investigation

  • The therapeutic potential of targeting LRRC55 requires further exploration

Addressing these research gaps would significantly advance our understanding of LRRC55 biology and its potential relevance to human health and disease.

How might LRRC55 be therapeutically targeted in disease conditions?

Based on current understanding of LRRC55 function, several potential therapeutic approaches could be considered:

• Gene Therapy Approaches:

  • Viral vector-mediated delivery of LRRC55 to enhance expression in target tissues

  • This approach might be particularly relevant for protecting pancreatic β-cells in diabetes

  • Adenoviral vectors have already demonstrated efficacy in experimental models

• Small Molecule Development:

  • Identification of compounds that enhance LRRC55 expression or function

  • High-throughput screening for molecules that mimic LRRC55's protective effects

  • Structure-based drug design targeting LRRC55 or its interaction partners

• Applications in Diabetes Therapy:

  • LRRC55 has been identified as "a potential therapeutic target in diabetes by reducing ER stress and promoting β-cell survival"

  • Strategies to upregulate or enhance LRRC55 could protect β-cells from stress-induced apoptosis

  • This could help preserve β-cell mass and function, addressing a fundamental aspect of diabetes pathophysiology

• Hormone-Based Approaches:

  • Given that LRRC55 expression is regulated by prolactin receptor signaling, prolactin or prolactin receptor agonists might indirectly enhance LRRC55 expression

  • This approach could leverage existing hormonal pathways to boost LRRC55-mediated protection

• Combination Therapy Strategies:

  • LRRC55-targeting approaches could be combined with other therapeutic modalities

  • For diabetes, this might include combination with agents that improve insulin sensitivity or reduce glucolipotoxicity

  • Such combinations might provide synergistic benefits by addressing multiple disease mechanisms

The therapeutic potential of LRRC55 is particularly promising in conditions involving ER stress and cellular apoptosis, though substantial research is still needed to translate current biological understanding into viable therapeutic strategies.