FKBPL Human

What is FKBPL and what protein family does it belong to?

FKBPL (FK506-binding protein like) is a member of the immunophilin protein superfamily with potent anti-tumor activity primarily through inhibition of angiogenesis and cancer stemness. Also known as DIR1, NG7, and WAF-1/CIP1 stabilizing protein 39 (WISp39), FKBPL contains 349 amino acids in humans and features a unique N-terminal region that confers its biological activity. Unlike some immunophilins, FKBPL contains a non-functional PPIase domain in this N-terminal region, which is independent of its TPR domains but critical for its biological effects .

What is the amino acid sequence of human FKBPL protein?

The full amino acid sequence of human FKBPL protein (1-349) is:
METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQI RQQPRDPPTETELELEVSPDPASQILEHTQGAEKLV AELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFV KKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEG WTELTMGVGPWEREETWGELIEKCLESMCQGEEA ELQLPGHSGPPVRLTLASFTQGRDSWELETSEKEA LAREERARGTELFRAGNPEGAARCYGRALRLLLTL PPPGPPERTV LHANLAACQLLLGQPQLAAQS CDRVLEREPGHLKALYRRGVAQAALGN LEKATAD LKKVLAIDPKNRAAQEELGKVVIQGKNQDAGLAQG LRKMF

Methodology note: When working with recombinant FKBPL, researchers often use the His-tagged version expressed in Escherichia coli with >90% purity, which is suitable for SDS-PAGE, mass spectrometry, and functional studies .

What are the known functions of FKBPL at the cellular level?

FKBPL has several important cellular functions:

• Regulation of vascular integrity through modulation of endothelial cell barrier function

• Negative regulation of NF-κB activation and inflammatory signaling

• Anti-angiogenic activity through CD44-dependent mechanisms

• Anti-cancer stem cell (CSC) activity by regulating expression of pluripotency transcription factors

• Regulation of p21 protein stability through binding to Hsp90 and p21

• Involvement in cellular response to X-ray radiation

When studying these functions, researchers should implement appropriate controls and utilize both gain-of-function and loss-of-function approaches to validate findings .

What are effective methods for FKBPL knockdown in cellular models?

For effective FKBPL knockdown, siRNA transfection has been successfully used in human microvascular endothelial cells (HMEC-1), achieving approximately 80% reduction in both mRNA and protein expression. This technique is particularly useful for investigating FKBPL's role in endothelial barrier function and inflammatory responses.

Methodological considerations:

• Validate knockdown efficiency by measuring both mRNA (qPCR) and protein (Western blot) levels

• Include appropriate non-targeted siRNA controls

• Consider the impact of inflammatory stimuli (e.g., LPS) on knockdown efficiency

• For phenotypic assays after knockdown, an in vitro Evan's blue permeability assay can effectively measure changes in endothelial barrier function .

How can researchers study FKBPL's role in NF-κB signaling?

To investigate FKBPL's role in NF-κB signaling:

• Isolate bone marrow-derived macrophages (BMDMs) from wild-type and Fkbpl+/- mice

• Stimulate cells with LPS (100 ng/ml) for different time points (e.g., 45, 60, 90 minutes)

• Assess phosphorylation of p65(RelA) via Western blotting

• For peptide studies, include AD-01 (1 nM) in treatment groups

• To determine CD44 dependency, use CD44 knockout mouse models

• Include controls for other inflammatory signaling pathways (e.g., JNK phosphorylation)

This approach allows for rigorous assessment of FKBPL's specific effects on NF-κB signaling while controlling for potential confounding effects on other inflammatory pathways .

What in vivo models are appropriate for FKBPL research?

Several in vivo models have proven valuable for FKBPL research:

• Fkbpl+/- haploinsufficient mice:

  • Useful for studying developmental angiogenesis

  • Show vascular irregularities and enhanced angiogenesis

  • Display increased erythrocyte leakage into surrounding tissues

  • Demonstrate reduced survival in LPS-induced inflammatory challenge

• LPS-induced inflammatory models:

  • Effective for testing FKBPL peptides (AD-01, ALM201) in vivo

  • Allow assessment of survival outcomes and inflammatory cytokine production

  • Permit collection of peritoneal lavage for cytokine analysis

• CD44 knockout mice (CD44-/-):

  • Valuable for determining CD44-dependency of FKBPL mechanisms

  • Can be used to differentiate between angiogenic and inflammatory pathways

When designing in vivo experiments, researchers should carefully consider dosing regimens, timing of interventions, and appropriate controls to account for genetic background effects .

How does FKBPL regulate vascular integrity in response to inflammatory stimuli?

FKBPL functions as a critical regulator of vascular integrity, particularly during inflammatory responses. When FKBPL is knocked down in human microvascular endothelial cells (HMEC-1), there is a significant increase in barrier permeability (p = 0.0347), which is further exacerbated by LPS stimulation. This indicates that FKBPL plays a protective role in maintaining endothelial barrier function.

At the molecular level, FKBPL regulates vascular integrity through:

• Modulation of VE-cadherin endothelial tight junctions

• Inhibition of NF-κB signaling in endothelial cells

• Regulation of inflammatory cytokine production

Importantly, treatment with the FKBPL-based peptide AD-01 increases VE-cadherin endothelial tight junctions following LPS stimulation, offering a potential therapeutic approach for stabilizing vascular barriers during inflammatory conditions .

What is the relationship between FKBPL and other immunophilins in regulating NF-κB?

FKBPL shares functional similarities with other immunophilins in regulating NF-κB signaling, but through distinct mechanisms:

• FKBP51 complexes with cytoplasmic p65 in unstimulated cells

• Upon stimulation, FKBP51 is exchanged for FKBP52

• FKBP52 is then recruited to the promoter region of NF-κB target genes

Important distinctions:

• The TPR domains of FKBP51 and FKBP52 are not required for NF-κB regulation

• FKBP52's PPIase activity is required for its NF-κB stimulatory function

• FKBP51's PPIase activity is not required for its inhibitory action

• FKBPL peptides (AD-01, ALM201) based on the N-terminal region (containing the non-functional PPIase domain) modulate NF-κB similarly to full-length FKBPL

These findings suggest that despite structural differences, FKBPL regulates NF-κB through its N-terminal domain, similar to but distinct from other immunophilins. Further research is needed to fully understand the interaction between FKBPL, FKBP51, and FKBP52 in NF-κB regulation .

How do genetic variations in FKBPL correlate with human disease phenotypes?

Genetic analysis has revealed significant associations between FKBPL variants and human inflammatory and autoimmune conditions:

Methodology for genetic association studies:

• Utilize global biobank engines comprising multiple geographical biobanks

• Assess variants in fkbpl, fkbp51, and fkbp52 associated with phenotypes of interest

• Apply genome-wide association study (GWAS) significance threshold of p≤ 5×10^-8

• Analyze tissue-specific gene expression levels using databases like GTEx

• Investigate disease-specific expression using microarray data from resources like NCBI GEO

Findings from such analyses have revealed associations between FKBPL genetic variants and:

• Psoriasis

• Rheumatoid arthritis

• High lymphocyte count

• Potentially other inflammatory disorders

These genetic associations support FKBPL's physiological role in regulating inflammatory responses and suggest genetic testing for FKBPL variants could help identify individuals at risk for inflammatory conditions .

What is the clinical significance of FKBPL-based peptides?

FKBPL-based peptides have shown remarkable potential as therapeutic agents:

• AD-01: A 24-residue peptide (amino acids 34-58 of FKBPL)

  • Pre-clinical peptide with potent anti-tumor activity

  • Inhibits p65(RelA) phosphorylation following LPS stimulation

  • Reduces NF-κB target gene expression and pro-inflammatory cytokine production

  • Functions independently of CD44 in regulating NF-κB signaling

• ALM201: A more stable 23-residue peptide

  • Clinical drug candidate with equipotent activity to AD-01

  • Completed Phase 1a dose-escalation clinical trial in patients with ovarian cancer and other solid tumors

  • Demonstrated favorable pharmacokinetic and safety profile with no major adverse events

  • Received orphan drug status from FDA for ovarian cancer

  • In LPS survival models, treatment resulted in 100% survival rate at experimental endpoint

  • Abrogated production of pro-inflammatory cytokines (TNF and IL-6) in peritoneal lavage washings

These peptides represent a significant advancement in developing FKBPL-based therapeutics that could address both vascular instability and excessive inflammation in conditions such as sepsis, acute respiratory distress syndrome (ARDS), and potentially severe COVID-19 .

How is FKBPL associated with metabolic disorders and cardiovascular disease?

Recent research has identified FKBPL as a potential biomarker and therapeutic target in metabolic and cardiovascular conditions:

A cross-sectional study of 353 adults (234 with Type 2 diabetes and 119 non-diabetic subjects) found:

• Higher plasma FKBPL levels in Type 2 diabetes patients (adjusted mean: 2.03 ng/ml ± 0.90 SD) compared to non-diabetic subjects (adjusted mean: 1.79 ng/ml ± 0.89 SD)

• Association between FKBPL levels and cardiovascular disease risk

These findings suggest:

• FKBPL may play a role in the pathophysiology linking diabetes and cardiovascular complications

• Disturbed angiogenesis and endothelial dysfunction, both regulated by FKBPL, are implicated in both conditions

• FKBPL could potentially serve as a biomarker for cardiovascular risk in diabetic patients

Researchers investigating FKBPL in metabolic disorders should employ appropriate case-control designs with matching for age, BMI, and gender to minimize confounding factors .

What therapeutic potential does FKBPL have in inflammatory conditions?

FKBPL-based therapeutics show promise for treating a range of inflammatory conditions:

• Acute inflammatory conditions:

  • Sepsis

  • Acute respiratory distress syndrome (ARDS)

  • Severe COVID-19

  • Other conditions characterized by cytokine storm

• Chronic inflammatory diseases:

  • Psoriasis

  • Rheumatoid arthritis

  • Potentially other autoimmune conditions

The dual activity of FKBPL-based peptides in:

• Stabilizing vascular barriers (reducing leakage)

• Inhibiting pro-inflammatory cytokine production

Makes them particularly valuable candidates for conditions where both vascular dysfunction and excessive inflammation contribute to pathology. The favorable safety profile demonstrated in clinical trials further enhances their therapeutic potential .

How should researchers approach contradictory findings in FKBPL research?

When faced with contradictory findings in FKBPL research, scientists should consider:

• Context-dependent effects:

  • FKBPL may have different functions in different cell types (e.g., endothelial cells vs. macrophages)

  • Inflammatory state may affect FKBPL's role (baseline vs. stimulated conditions)

  • Interaction with different binding partners in different tissues

• Methodological differences:

  • In vitro vs. in vivo models

  • Acute vs. chronic interventions

  • Genetic models vs. pharmacological approaches

  • Differences in experimental conditions (e.g., LPS concentration, timing)

• Experimental approaches to resolve contradictions:

  • Use multiple complementary techniques to study the same phenomenon

  • Include appropriate positive and negative controls

  • Validate findings across different cell types and experimental models

  • Consider dose-response relationships rather than single-dose experiments

  • Perform time-course studies to capture dynamic changes

By systematically addressing these factors, researchers can better understand the complex and context-dependent functions of FKBPL .

What are the optimal methods for measuring FKBPL in human samples?

For accurate measurement of FKBPL in human samples:

• Plasma/Serum quantification:

  • ELISA has been successfully used to measure FKBPL levels in plasma from diabetic and non-diabetic subjects

  • Important to standardize collection, processing, and storage of samples

• Tissue expression:

  • Immunohistochemistry can assess FKBPL expression in tissue samples

  • qPCR for mRNA quantification

  • Western blotting for protein expression

• Genetic analysis:

  • Genotyping of FKBPL variants using next-generation sequencing

  • Analysis of expression quantitative trait loci (eQTLs) using databases like GTEx

• Methodological considerations:

  • Include appropriate controls for each assay

  • Account for potential confounding factors (age, gender, BMI, medications)

  • Validate findings using multiple methodological approaches

  • Consider tissue-specific expression patterns when interpreting results

Researchers should select methods based on their specific research questions and available sample types .