Differences in signal activation by LH and hCG are mediated by the LH/CG receptor’s extracellular hinge region

The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) – secreted by the placenta, and lutropin (LH) – produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor’s leucine-rich- repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor

Age-related Changes in Human Bone Proteoglycan Structure: IMPACT OF OSTEOGENESIS IMPERFECTA

Proteoglycans (PGs) are a family of molecules that undergo extensive post-translational modifications that include addition of glycosaminoglycan (GAG) chains as well as N- and O-linked oligosaccharides to the protein core. PG composition and structure have been reported to alter with age. To test whether the post-translational modifications to PGs can serve as in vitro surrogate end point markers for chronological age, the extent of GAG modifications was determined for PGs derived from normal human bone cells of 14 donors (age range, fetal to 60 years). Isolated cells were steady state radiolabeled with (35)SO(4)(2-) and [(3)H]GlcN. For biglycan and decorin, iduronate content was linearly correlated with age (increased 1.5x between fetal and age 60 years). For the syndecan-like heparan sulfate PG, the N-sulfation of post-natal cells increased over 3.5-fold until reaching a plateau during the 4th decade of life. The amount of O-linked oligosaccharides was also found to decrease as a function of increasing normal donor age, whereas the specific activity of the metabolic precursor pool remained constant regardless of donor age. These age-related changes in post-translational modifications were then used to demonstrate that osteoblasts derived from patients with osteogenesis imperfecta did not exhibit facets of a pre-mature aging, but rather were arrested in a fetal-like phenotypic state. A growth matrix rich in thrombospondin altered PG metabolism in osteoblastic cells, resulting in the production and secretion of the fetal-like (rich in O-linked oligosaccharides) forms of decorin and biglycan. This effect was qualitatively different from the effect of transforming growth factor-beta, which predominantly altered GAGs rather than O-linked oligosaccharides. No other Arg-Gly-Asp protein (fibronectin, vitronectin, type I collagen, osteopontin, and bone sialoprotein) showed any detectable effect on PG metabolism in bone cells. These results indicate that a proper matrix stoichiometry is critical for metabolism of PGs

Age-Related Adaptation of Bone-PDL-Tooth Complex: Rattus-Norvegicus as a Model System

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7±0.1 to 0.9±0.2 GPa) and cementum (0.6±0.1 to 0.8±0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated

Structure-function studies of the hinge region in glycoprotein hormone receptors

Zusammen mit dem Thyreotropinrezeptor (TSHR), bilden der Follitropinrezeptor (FSHR) und der Lutropinrezeptor (LHR) die Untergruppe der Glykoproteinhormon- Rezeptoren (GPHR), welche zu den rhodopsinähnlichen GPCR zählt. Die GPHR bilden eine oligomere Signalisierungseinheit, bei der die Signalweiterleitung durch intermolekulare Interaktionen mit benachbarten Rezeptoren und endogenen Liganden erfolgt. Eine Beteiligung an diesem Prozess wird von der Hinge-Region vermutet, welche die extrazelluläre ligandbindenden leucine-rich repeat Domäne (LRRD) mit der transmembranären Serpentindomäne (SD) verbindet. Um den molekularen Mechanismus der Signalvermittlung zu verstehen, wurde in der vorliegenden Arbeit erstmalig das strukturelle Profil der Hinge-Region des TSHR und des LHR untersucht. Mittels breit angelegter Expressionsstudien in E.coli und P.pastoris, sowie einem proteinfragmentbasiertem Hochdurchsatz- Screening (ESPRIT) konnte gezeigt werden, dass die Hinge-Region funktionell essentielle strukturierte Abschnitte aufweist, diese jedoch aber nicht strukturautonom als Domäne vorliegen. Vielmehr führen intra- und intermolekulare Kontakte von benachbarten Domänen zur Ausbildung aktiver Konformationen, welche zur Signalamplifikation führen. Weiterhin konnten durch die Einführung von Alanin bzw. Prolinmutationen in den Exon10-kodierenden Bereich der Hinge-Region zwei potentiell helikale Strukturelemente, sowie eine signalisierungssensitive Region innerhalb der LHR Hinge-Region gefunden werden, welche in unterschiedlicher Weise mit den Hormonen Lutropin (LH) und Choriongonadotropin (CG) interagieren. Mit Hilfe von Mutationen und Deletionen innerhalb der LHR Hinge-Region konnte desweiteren gezeigt werden, dass nicht spezifische Aminosäureeigenschaften, sondern vielmehr Strukturmerkmale funktionsrelevant sind und im Fall einer strukturellen Störung (Deletion) Funktionen im Signalisierungsmechanismus kompensiert werden. Für den LHR konnte zum ersten Mal gezeigt werden, dass für die beiden Hormone LH und CG unterschiedliche molekulare Aktivierungsmechanismen im Zusammenhang mit der Hinge-Region vorliegen. Dabei wurde die intramolekulare Aktivierung durch LH- und die intermolekulare Aktivierung durch CG-Stimulation nachgewiesen. Die beleuchteten molekularen Determinanten für die Rezeptoraktivierung liefern ebenfalls eine plausible Erklärung für die Fehlfunktion einer natürlich vorkommenden Deletionsmutante bei der die LHR Hinge-Region um 27 Aminosauren (Exon10) verkürzt ist. Diese Arbeit zur Hinge-Region der GPHR bietet somit eine Grundlage für die Formulierung eines erweiterten Aktivierungsmodells für den LHR unter Einbezug kooperativer Effekte und beschreibt zum ersten Mal den Einfluss der Hinge-Region in dem ablaufenden Aktivierungsprozess.Together with the thyroid stimulating hormone receptor (TSHR), the follitropin receptor (FSHR) and the lutropin receptor (LHR) constitutes the subgroup of glycoprotein-hormone receptors (GPHR), which belongs to the rhodopsin like GPCR. The GPHR act as oligomeric signaling units in which signal transduction is mediated by intermolecular interaction of neighboring receptors and endogenous ligands. It is speculated that the intermolecular cooperativity is mainly influenced by the hinge region, a key participant for signal transduction connecting the extracellular ligand binding leucine-rich repeat domain (LRRD) and the transmembrane spanning region (SD). This work addresses the role of the hinge region in this process by elucidation of the structure- function relationship during the signal initiation and transduction. One goal of this work is the exploration of its structural profile. To reveal insights in the folding properties of extracellular TSHR fragments, different expression strategies (E. coli and P. pastoris) has been performed. For extensive expression studies a DNA library based high-throughput screen (ESPRIT) was performed. TSHR hinge region fragments with an enhanced stability could be prepared for further structural investigations. Initial experiments revealed that the hinge region cannot be isolated as an autonomously self- folding domain. It is likely that the hinge region needs additional parameters like tight intra- or intermolecular contact partners to achieve the correct fold. A second goal is to reveal the molecular reasons for the different signaling behavior of lutropin (LH) and choriongonadotropin (CG) at the lutropin receptor (LHR). To elucidate the structure-function relationship a LHR mutant lacking 27 amino acids (exon10) within the hinge region was investigated. Since proline but not alanine mutations impair the LHR function it is causative that signal transduction is mediated rather by two identified structural elements than by specific amino acid motifs. Moreover it could be shown that LH mediated receptor activation is strongly related to a sulfated tyrosine within the Tyr331 region, while CG activation is less dependent and activates the receptor by interaction with the structural element within exon10 of the LHR hinge region. A third goal of this work is the elucidation of the differences between LH and CG induced function by investigation of intermolecular cooperativity at the LHR. For this purpose the reported LHR mutant lacking exon10 has been investigated in an experimental setup, in which the combination of signal deficient LHR mutants and hormone binding deficient ones were tested for intermolecular receptor activation. Fluorescence cross- correlation spectroscopy (FCCS) excluded monomerization of LHR-delEx10 as reason for functional differences. Moreover, it could be shown that LH may exclusively stimulate the targeted LHR by intramolecular activation, while CG is able to induce intermolecular activation too

The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment

Cryogenic cooling-based sustainable machining

Machinability is the most important indicator to measure the performance of any machining operation. Cutting force, surface roughness, tool wear, dimensional accuracy, and cutting temperature, etc. are the most important machinability indicators. Various materials such as inconel, titanium, haynes, and waspalloy, etc. are difficult-to-machine (DTM) materials. Cryogenic cooling is one of the techniques to improve the machinability of these materials, and found effective specially when machining at high speed. This chapter sheds light on cryogenic cooling-based machining, its working principle and mechanism, and case studies on cryogenic cooling-based machining of various DTM materials. © 2020 Elsevier Inc. All rights reserved