Protective hinge in insulin opens to enable its receptor engagement

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated “microreceptors” that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe^B24 to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor''s L1–β₂ sheet. Opening of this hinge enables conserved nonpolar side chains (Ile^A2, Val^A3, Val^B12, Phe^B24, and Phe^B25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.How insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24–B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3–6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residue-specific photo–cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ)₂ receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1^*) comprises the central β-sheet (L1–β₂) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical C-terminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits’ intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently described at maximum resolution of 3.9 Å (11), revealing that hormone binding is primarily mediated by αCT (receptor residues 704–719); direct interactions between insulin and L1 were sparse and restricted to certain B-chain residues. On insulin binding, αCT was repositioned on the L1–β₂ surface, and its helix was C-terminally extended to include residues 711–714. None of these structures defined the positions of C-terminal B-chain residues beyond B21. Support for the detachment model was nonetheless provided by entry of αCT into a volume that would otherwise be occupied by B-chain residues B25–B30 (i.e., in classical insulin structures; Fig. 1B) (11).Open in a separate windowFig. 1.Insulin B-chain C-terminal β-strand in the μIR complex. (A) Structure of apo-receptor ectodomain. One monomer is in tube representation (labeled), the second is in surface representation. L1, first leucine-rich repeat domain; CR, cysteine-rich domain; L2, second leucine-rich repeat domain; FnIII-1, -2 and -3; first, second and third fibronectin type III domains, respectively; αCT, α-subunit C-terminal segment; coral disk, plasma membrane. (B) Insulin bound to μIR; the view direction with respect to L1 in the apo-ectodomain is indicated by the arrow in A. Only B-chain residues indicated in black were originally resolved (11). The brown tube indicates classical location of residues B20-B30 in free insulin, occluded in the complex by αCT. (C) Orthogonal views of unmodeled 2F_obs-F_calc difference electron density (SI Appendix), indicating association of map segments with the αCT C-terminal extension (transparent magenta), insulin B-chain C-terminal segment (transparent gray), and Asn^A21 (transparent yellow). Difference density is sharpened (B_sharp = −160 Ų). (D–F) Refined models of respective segments insulin B20–B27, αCT 714–719, and insulin A17-A21 within postrefinement 2F_obs-F_calc difference electron density (B_sharp = −160 Ų). D is in stereo.We describe here the structure and interactions of the detached B-chain C-terminal segment of insulin on its binding to a “microreceptor” (μIR), an L1–CR domain-minimized version of the α-subunit (designated IR310.T) plus exogenous αCT peptide 704–719 (11). Our analysis defines a hinge in the B chain whose opening is coupled to repositioning of αCT between nonpolar surfaces of L1 and the insulin A chain. To understand the role of this hinge in holoreceptor binding and signaling, we designed three insulin analogs containing structural constraints (d-Ala^B20, d-Ala^B23]-insulin, ∆Phe^B25-insulin, and ∆Phe^B24-insulin, where ∆Phe is (α,β)-dehydrophenylalanine (Fig. 2) (12). The latter represents, to our knowledge, the first use of ∆Phe—a rigid “β-breaker” with extended electronic conjugation between its side chain and main chain (SI Appendix, Fig. S1)—as a probe of induced fit in macromolecular recognition. In addition, a fourth analog, active but with anomalous flexibility in the B chain (5, 6) (

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.     

Coronavirus Disease-2019 (COVID-19) and the Liver

Within a year of its emergence, coronavirus disease-2019 (COVID-19) has evolved into a pandemic. What has emerged during the past 1 year is that, apart from its potentially fatal respiratory presentation from which the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) derives its name, it presents with a myriad of gastrointestinal (GI) and liver manifestations. Expression of the angiotensin-converting enzyme-2 (ACE-2) receptor throughout the GI tract and liver, which is the receptor for the SARS-CoV-2, may be responsible for the GI and liver manifestations. Besides acting directly via the ACE-2 receptor, the virus triggers a potent immune response, which might have a role in pathogenesis. The virus leads to derangement in liver function tests in close to 50% of the patients. The impact of these derangements in patients with a normal underlying liver seems to be innocuous. Severe clinical presentations include acute decompensation and acute-on-chronic liver failure in a patient with chronic liver disease, leading to high mortality. Evolving data suggests that, contrary to intuition, liver transplant recipients and patients with autoimmune liver disease on immunosuppression do not have increased mortality. The exact mechanism underlying why immunosuppressed patients fare well as compared to other patients remains to be deciphered. With newer variants of COVID-19, which can spread faster than the original strain, the data on hepatic manifestations needs to be updated to keep a step ahead of the virus.     

Magnetic resonance cholangiopancreatography in obstructive jaundice

GOALS: To determine the ability of magnetic resonance cholangiopancreatography (MRCP) to diagnose the level and cause of obstruction in patients with obstructive jaundice. BACKGROUND: The limitations of available imaging modalities have led to the increasing use of MRCP, which is a noninvasive and highly accurate technique in evaluating patients with biliary obstruction. STUDY: Thirty patients were included in this study. MRCP was done using a fat suppressed, heavily T2 weighted fast spin echo sequence. The MRCP findings were confirmed on surgical exploration or clinical follow-up. RESULTS: MRCP could correctly identify ductal dilatation and the level of obstruction in all cases, except one. All causes of obstruction, except three, were detected. It failed to detect a common bile duct calculus in a minimally dilated ductal system and misdiagnosed a case of focal chronic pancreatitis as carcinoma head pancreas and a small pancreatic head mass as cholangiocarcinoma. It had a sensitivity of 94.44%, specificity of 81.81%, positive predictive value of 89.47%, and negative predictive value of 90% for the detection of malignant causes. The overall diagnostic accuracy for detection of level and cause of obstruction was 96.3% and 89.65%, respectively. CONCLUSION: The high diagnostic accuracy of MRCP in evaluating patients with obstructive jaundice indicates that it has the potential to become the diagnostic modality of choice in such patients.     

The evidence for abandoning the amniotic fluid index in favor of the single deepest pocket

This study assessed whether the amniotic fluid index (AFI) or the single deepest pocket (SDP) is the best technique to estimate amniotic fluid volume. The AFI and SDP were compared to a dye-determined or directly measured amniotic fluid volume. A PUBMED search from 1990 to 2006 was conducted using the search terms "single deepest pocket" or "largest vertical pocket" or "maximum vertical pocket" or "2X1 pocket" AND "amniotic fluid index". One study compared the AFI and SDP to a dye-determined amniotic fluid volume. There were 1219 publications that used the search term SDP-LVP-MVP versus 4378 using AFI. Twenty publications contained both the AFI and SDP, but only six compared the AFI and SDP. Both the AFI and the SDP poorly identified abnormal amniotic fluid volumes, and neither technique was superior to the other. The AFI identifies a significantly greater number of women as having oligohydramnios versus the SDP but without any difference in perinatal outcomes. Compared with SDP, AFI excessively characterizes a greater number of pregnancies as having oligohydramnios leading to more interventions without improvement in perinatal outcome. The AFI should be abandoned and the SDP used to estimate amniotic fluid volume.     

Amniotic fluid index as a predictor of adverse perinatal outcome in the HELLP syndrome

OBJECTIVE: To evaluate the prognostic value of an amniotic fluid index (AFI) < or = 5 cm for an adverse perinatal outcome in pregnancies with the syndrome of hemolysis, elevated liver enzymes and low platelets (HELLP syndrome). STUDY DESIGN: A prospective, observational study of patients with the HELLP syndrome. An ultrasound estimate of amniotic fluid volume was obtained on admission. Adverse intrapartum outcomes included amnioinfusion for variable decelerations and/or indicated abdominal/vaginal operative delivery for nonreassuring fetal heart rate changes. Maternal characteristics and perinatal outcome parameters were compared AFI < or = vs. > 5 cm. Statistical analysis was performed using chi2 analysis, Student's t test and receiver-operator characteristic curve (ROC) analysis. RESULTS: Between January 1996 and February 1999, 120 patients were enrolled. Twenty-six (22%) had an AFI < or = 5 cm. This group did not differ from that with AFI > 5 cm regarding the severity of the HELLP syndrome, admission-to-delivery interval (p = 0.354), variable decelerations in labor (p = 0.06), Apgar score of < 7 at 5 minutes (p = 0.361), cesarean delivery for nonreassuring fetal status (p = 1.0) or significant fetal acidosis (pH < 7.0 [p = 0.2101). ROC analysis revealed no AFI measurement between 0 and 16 cm that was useful for identifying the compromised fetus. CONCLUSION: Antepartum/intrapartum performance of AFI in patients with the HELLP syndrome is a poor prognostic test for subsequent fetal compromise.     

Role of CLEC-2-driven platelet activation in the pathogenesis of toxic liver damage

Background

Toxic liver injury from drugs including paracetamol is the main cause of acute liver failure in developed countries. The mechanisms that drive irreversible liver failure are poorly understood; platelets could have an important role in this process given their roles beyond haemostasis, including liver regeneration. Ligation of the platelet receptor CLEC-2 with its cognate ligand podoplanin (PDPN) powerfully activates platelets; we sought to investigate the role of CLEC-2 in the pathogenesis of acute liver failure.