Urinary Kringle Domain-Containing Protein HGFL: A Validated Biomarker of Early Sickle Cell Anemia-Associated Kidney Disease

<b><i>Introduction:</i></b> Chronic kidney disease (CKD) is a prevalent complication of sickle cell anemia (SCA). Hyperfiltration that delayed detection of CKD is common in SCA patients. Identification of novel urinary biomarkers correlating with glomerular filtration rates may help to detect and predict progression of renal disease. <b><i>Methods:</i></b> Reanalysis of mass spectra of urinary samples obtained from University of Illinois at Chicago identified kringle domain-containing protein HGFL. <b><i>Results:</i></b> HGFL levels correlated with hyperfiltration, were significantly reduced at CKD stage 1 compared to stage 0, negatively correlated with progression of CKD and were suitable for differentiation of stage 1. Better prediction of CKD progression to stage 2 was observed for HGFL-based risk prediction compared to the estimated glomerular filtration rate (eGFR)-based prediction. Results from a Howard University patient cohort supported the utility of HGFL-based test for the differentiation of stage 1 of CKD. <b><i>Conclusion:</i></b> Urinary HGFL may contribute additional information beyond eGFR and improve diagnosis of early-stage CKD in SCA patients.

The constitutive high-affinity Met-binding site in the kringle domain is dispensable for the signalling activity of hepatocyte growth factor

Activation of a tyrosine kinase receptor Met by hepatocyte growth factor (HGF) requires binding of proteolytically activated, two-chain (tc) HGF, but the biochemical detail of this ligand–receptor interaction specificity remains elusive because biologically inactive single chain (sc) HGF can also bind to Met with high affinity. We found that this proteolysis-independent Met binding can be eliminated by mutagenesis introduced in the kringle domain without losing the ability to bind and activate cellular Met receptor after proteolytic activation, arguing against this site’s involvement in the physiological signalling. This non-signal producing Met–HGF interaction can also be eliminated by addition of a heparin mimetic sucrose octasulphate (SOS). By including SOS in the running buffer, we succeeded in detecting cleavage-dependent tcHGF–Met complex formation by size exclusion chromatography.

PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt

Phosphatidylinositol-3 kinases (PI3Ks) modulate cellular growth, proliferation, and survival; dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or transmembrane inhibitor of PI3K [TrIP]) is a putative transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular domain with homology to the inter-SH2 domain of the PI3K regulatory subunit p85, but the mechanism of TrIP function is poorly understood. We show that both the kringle and p85-like domains are necessary for TrIP inhibition of PI3K and that TrIP is down-modulated from the surface of T cells during T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by mediating oligomerization. Using an inducible knockout mouse model, we show that TrIP-deficient T cells exhibit more robust activation and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is a negative regulator of T cell activation and may represent a novel target for immune modulation.

PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt

ABSTRACTPhosphatidylinositol-3 kinases (PI3Ks) modulate numerous cellular functions, including growth, proliferation and survival. Dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or Transmembrane Inhibitor of PI3K – TrIP) is a novel transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular “p85-like” domain with homology to the inter-SH2 domain of the regulatory subunit of PI3K. Although TrIP has been shown to bind to the p110 catalytic subunit of PI3K in fibroblasts, the mechanism by which TrIP functions is poorly understood. We show that both the kringle and “p85-like” domains are necessary for TrIP inhibition of PI3K. We also demonstrate that TrIP protein is down-modulated from the surface of T cells to allow T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by binding an as-yet-unidentified ligand. Using an inducible knockout mouse model that we developed, we show that TrIP-deficient T cells exhibit more robust T cell activation, show a preference for Th1 polarization and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is an important negative regulator of T cell activation and may represent a novel target for immune modulation therapies.

Exploring the chemical space of the lysine-binding pocket of the first kringle domain of hepatocyte growth factor/scatter factor (HGF/SF) yields a new class of inhibitors of HGF/SF-MET binding

Targeting thelysine-binding pocketof the first kringle domain of HGF/SF using a fragment-based approach identified new chemical entities that can inhibit MET signalling.