The Unique Cysteine Knot Regulates the Pleotropic Hormone Leptin

Leptin plays a key role in regulating energy intake/expenditure, metabolism and hypertension. It folds into a four-helix bundle that binds to the extracellular receptor to initiate signaling. Our work on leptin revealed a hidden complexity in the formation of a previously un-described, cysteine-knotted topology in leptin. We hypothesized that this unique topology could offer new mechanisms in regulating the protein activity. A combination of in silico simulation and in vitro experiments was used to probe the role of the knotted topology introduced by the disulphide-bridge on leptin folding and function. Our results surprisingly show that the free energy landscape is conserved between knotted and unknotted protein, however the additional complexity added by the knot formation is structurally important. Native state analyses led to the discovery that the disulphide-bond plays an important role in receptor binding and thus mediate biological activity by local motions on distal receptor-binding sites, far removed from the disulphide-bridge. Thus, the disulphide-bridge appears to function as a point of tension that allows dissipation of stress at a distance in leptin.     

Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains.

The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V(32), and F(37)) adopting consistent interspatial positions with respect to one another. Peptides in the P(34) series were comparatively more flexible at the C-terminus, although a large proportion of the [D(31),P(34),F(35)]CGRP(27-37)-NH(2) calculated conformers contained a gamma-turn centered on P(34). These results collectively suggest that turn structures at both the C-terminus and N-terminus of CGRP(27-37)-NH(2) analogues may help to appropriately orient critical residues (T(30), V(32), and F(37)) for hCGRP(1) receptor binding.     

The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors

The small leucine-rich repeat proteins, fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-sulfated tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N and C termini, respectively, and PRELP is known to bind to heparin via this domain. Another heparin-binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C terminus. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full-length osteoadherin. The tyrosine sulfate domains also bound heparin-binding proteins such as basic fibroblast growth factor-2, thrombospondin I, MMP13, the NC4 domain of collagen IX, and interleukin-10. Fibronectin with large heparin-binding domains did not bind, neither did CILP containing a heparin-binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the sulfated tyrosine residues shown by different binding properties of 10-kDa fragments subfractionated by ion-exchange chromatography. These interactions may sequester growth factors, cytokines, and matrix metalloproteinases in the extracellular matrix as well as contribute to its organization.The integrity of the extracellular matrix depends on a multitude of interactions between molecular constituents leading to the formation of major macromolecular assemblies important for tissue functions. A major component in most types of extracellular matrix is the network of fibrillar structures primarily composed of collagen I in fibrous tissues and bone, whereas cartilage contains the similar collagen II.These collagen fibrils contain a number of associated molecules, often bound to their surface. One such molecule is the distinct collagen IX, containing three triple helical domains each surrounded by non-triple helical domains. Another set of molecules binding to triple helical collagen is the members of the small leucine-rich repeat protein family, such as fibromodulin (1), lumican (2), decorin (3), biglycan (4), PRELP (5), chondroadherin (6), and possibly osteoadherin. The typical LRR³ protein contains 10–11 repeats of some 25 amino acids with leucine residues at conserved locations. This domain represents a common denominator for the family and contains structures providing for interaction with, e.g. triple helical collagen (7–9). The LRR proteins contain an extension at either the N- or C-terminal end or, in a few cases, at both ends. These extensions may contribute to a second function exemplified by PRELP, where the N-terminal with a stretch of clustered arginine residues provides binding to heparin/heparan sulfate containing optimally five or more disaccharides with three sulfate groups each (10). In decorin and biglycan, the N-terminal extension have substituents of glycosaminoglycan chains of dermatan/chondroitin sulfate that can contribute to collagen binding (11) as well as provide putative self interactions with a similar chain on another molecule. In particular, it has been shown that decorin and biglycan will bind via their protein core to the N-terminal globular domain of collagen VI (4) and direct the formation of the collagen VI-beaded filament network, provided that the glycosaminoglycan chains are intact.There are a number of proteins known to interact with heparin. Whereas heparin is not present in the extracellular matrix, these proteins may bind to stretches within the heparan sulfate chains enriched in disaccharides having high sulfate content. The heparan sulfate is found particularly as a component of cell surface proteoglycans such as glypicans (12) and syndecans (13) and of the extracellular matrix proteoglycans perlecan (14) and agrin (15). Important ligands for these chains are growth factors exemplified by members of the FGF family. Other molecules that bind to heparan sulfate include fibronectin, having two such domains with molecular weights of around 20,000 (16). Also several members of the metalloproteinase family contain heparin-binding motifs as do many cytokines.The most common heparin-binding sequence contains clusters of basic amino acids, often with additional proline residues. PRELP and chondroadherin as well as the other proteins mentioned represent examples having such sequences. A different type of motif, first found in thrombospondin I, contains consecutive repeats of a WXZ sequence, where the tryptophan may be mannosylated (17, 18). This is referred to as the thrombospondin type I motif heparin-binding structure. Thrombospondin I in addition contains a heparin-binding basic cluster of amino acids (19). CILP on the other hand only contains the thrombospondin type 1 motif. These domains can bind to heparin with high affinity, an interaction that can be disrupted by high salt.A very different type of extension is found in the N-terminal part of fibromodulin and osteoadherin. These proteins contain a number of tyrosine residues, which may and often do, carry a sulfate group. Thus, fibromodulin contains up to nine such residues and osteoadherin as many as eight, where six are located in the N-terminal and two in the C-terminal extension (20). Any given preparation contains molecules within the same species with a range of levels of O-sulfate-substituted tyrosine. The functional significances of these domains have been unknown. We now show that these domains can mimic heparin in several interactions.     

Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.     

Attempted Suicide in Bipolar Disorder: Risk Factors in a Cohort of 6086 Patients

Objective

Bipolar disorder is associated with high risk of self-harm and suicide. We wanted to investigate risk factors for attempted suicide in bipolar patients.

Method

This was a cohort study of 6086 bipolar patients (60% women) registered in the Swedish National Quality Register for Bipolar Disorder 2004–2011 and followed-up annually 2005–2012. Logistic regression was used to calculate adjusted odds ratios for fatal or non-fatal attempted suicide during follow-up.

Results

Recent affective episodes predicted attempted suicide during follow-up (men: odds ratio = 3.63, 95% CI = 1.76–7.51; women: odds ratio = 2.81, 95% CI = 1.78–4.44), as did previous suicide attempts (men: odds ratio = 3.93, 95% CI = 2.48–6.24; women: odds ratio = 4.24, 95% CI = 3.06–5.88) and recent psychiatric inpatient care (men: odds ratio = 3.57, 95% CI = 1.59–8,01; women: odds ratio = 2.68, 95% CI = 1.60–4.50). Further, those with many lifetime depressive episodes were more likely to attempt suicide. Comorbid substance use disorder was a predictor in men; many lifetime mixed episodes, early onset of mental disorder, personality disorder, and social problems related to the primary group were predictors in women.

Conclusion

The principal clinical implication of the present study is to pay attention to the risk of suicidal behaviour in bipolar patients with depressive features and more severe or unstable forms of the disorder.