Exploring the folding landscape of leptin: Insights into threading pathways.

The discovery of new protein topologies with entanglements and loop-crossings have shown the impact of local amino acid arrangement and global three-dimensional structures. This phenomenon plays a crucial role in understanding how protein structure relates to folding and function, affecting the global stability, and biological activity. Protein entanglements encompassing knots and non-trivial topologies add complexity to their folding free energy landscapes. However, the initial native contacts driving the threading event for entangled proteins remains elusive. The Pierced Lasso Topology (PLT) represents an entangled topology where a covalent linker creates a loop in which the polypeptide backbone is threaded through. Compared to true knotted topologies, PLTs are simpler topologies where the covalent-loop persists in all conformations. In this work, the PLT protein leptin, is used to visualize and differentiate the preference for slipknotting over plugging transition pathways along the folding route. We utilize the Energy Landscape Visualization Method (ELViM), a multidimensional projection technique, to visualize and distinguish early threaded conformations that cannot be observed in an in vitro experiment. Critical contacts for the leptin threading mechanisms were identified where the competing pathways are determined by the formation of a hairpin loop in the unfolded basin. Thus, prohibiting the dominant slipknotting pathway. Furthermore, ELViM offers insights into distinct folding pathways associated with slipknotting and plugging providing a novel tool for de novo design and in vitro experiments with residue specific information of threading events in silico.

The solution structure of human leptin reveals a conformational plasticity important for receptor recognition.

Leptin is a multi-potency cytokine that regulates various physiological functions, including weight control and energy homeostasis. Signaling of leptin is also important in many aging-related diseases. Leptin is required for the noncovalent crosslinking of different extracellular domains of leptin receptors, which is critical for receptor activation and downstream signaling. Nevertheless, the structure of intact apo-form leptin and the structural transition leptin undergoes upon receptor binding are not fully understood yet. Here, we determined the monomeric structure of wild-type human leptin by solution-state nuclear magnetic resonance spectroscopy. Leptin contains an intrinsically disordered region (IDR) in the internal A-B loop and the flexible helix E in the C-D loop, both of which undergo substantial local structural changes when leptin binds to its receptor. Our findings provide further insights into the molecular mechanisms of leptin signaling.

Topological Reaction Coordinate Captures the Folding Transition State Ensemble in a Pierced Lasso Protein.

Proteins with a pierced lasso topology (PLT) have a covalent loop created by a disulfide bond, and the backbone circles back to thread the loop. This threaded topology has unique features compared to knotted topologies; notably, the topology is controlled by the chemical environment and the covalent loop remains intact even when denatured. In this work, we use the hormone leptin as our model PLT system and study its folding using molecular dynamics simulations that employ a structure-based (Go̅-like) model. We find that the reduced protein has a two-state folding mechanism with a transition state ensemble (TSE) that can be characterized by the reaction coordinate Q, the fraction of native contacts formed. In contrast, the oxidized protein, which must thread part of the polypeptide chain through a covalent loop, has a folding process that is poorly characterized by Q. Instead, we find that a topological coordinate that monitors the residue crossing the loop can identify the TSE of oxidized leptin. By precisely identifying the predicted TSE, one may now reliably calculate theoretical phi-values for the PLT protein, thereby enabling a comparison with experimental measurements. We find the loop-threading constraint leads to noncanonical phi-values that are uniformly small because this PLT protein has a flat energy landscape through the TSE.

A Small Contribution to a Large System: The Leptin Receptor Complex.

Obesity is a classified epidemic, increasing the risk of secondary diseases such as diabetes, inflammation, cardiovascular disease, and cancer. The pleiotropic hormone leptin is the proposed link for the gut-brain axis controlling nutritional status and energy expenditure. Research into leptin signaling provides great promise toward discovering therapeutics for obesity and its related diseases targeting leptin and its cognate leptin receptor (LEP-R). The molecular basis underlying the human leptin receptor complex assembly remains obscure, due to the lack of structural information regarding the biologically active complex. In this work, we investigate the proposed receptor binding sites in human leptin utilizing designed antagonist proteins combined with AlphaFold predictions. Our results show that binding site I has a more intricate role in the active signaling complex than previously described. We hypothesize that the hydrophobic patch in this region engages a third receptor forming a higher-order complex, or a new LEP-R binding site inducing allosteric rearrangement.

Atomistic mechanism of leptin and leptin-receptor association.

The leptin-leptin receptor complex is at the very core of energy homeostasis and immune system regulation, among many other functions. In this work, we built homology models of leptin and the leptin binding domain (LBD) of the receptor from humans and mice. Docking analyses were used to obtain the coordinates of the native leptin-LBD complexes and a mixed heterodimer formed by human leptin and mouse LBD. Molecular dynamics (MD) simulations were performed using all models (monomers and heterodimers) as initial coordinates and the GROMACS program. The overall structural and dynamical behaviors are similar for the three complexes. Upon MD simulations, several new interactions appear. In particular, hydrophobic interactions, with more than 90% persistence, seem to be the most relevant for the stability of the dimers, as well as the pair formed by Asp85Lep and Arg468LBD. This in silico analysis provides structural and dynamical information, at the atomistic level, about the mechanism of leptin-LBD complex formation and leptin receptor activation. This knowledge might be used in the rational drug design of therapeutics to modulate leptin signaling.Communicated by Ramaswamy H. Sarma.

Leptin-Activity Modulators and Their Potential Pharmaceutical Applications.

Leptin, a multifunctional hormone primarily, but not exclusively, secreted in adipose tissue, is implicated in a wide range of biological functions that control different processes, such as the regulation of body weight and energy expenditure, reproductive function, immune response, and bone metabolism. In addition, leptin can exert angiogenic and mitogenic actions in peripheral organs. Leptin biological activities are greatly related to its interaction with the leptin receptor. Both leptin excess and leptin deficiency, as well as leptin resistance, are correlated with different human pathologies, such as autoimmune diseases and cancers, making leptin and leptin receptor important drug targets. The development of leptin signaling modulators represents a promising strategy for the treatment of cancers and other leptin-related diseases. In the present manuscript, we provide an update review about leptin-activity modulators, comprising leptin mutants, peptide-based leptin modulators, as well as leptin and leptin receptor specific monoclonal antibodies and nanobodies.

Specific Binding of Leptin to Red Blood Cells Delivers a Pancreatic Hormone and Stimulates ATP Release.

Since its discovery in 1994, leptin continues to have new potential physiological roles uncovered, including a role in the regulation of blood flow. Leptin's role in regulating blood flow is not completely understood. Red blood cell (RBC)-derived ATP is a recognized stimulus of blood flow, and multiple studies suggest that C-peptide, a hormone secreted in equimolar amounts with insulin from the pancreatic ß-cells, can stimulate that release when delivered by albumin and in combination with Zn2+. Here, we report leptin delivers C-peptide and Zn2+ to RBCs in a saturable and specific manner. We labeled leptin with technetium-99 m (99mTc) to perform binding studies while using albumin to block the specific binding of 99mTc-leptin in the presence or absence of C-peptide. Our results suggest that leptin has a saturable and specific binding site on the RBC ((Kd = 1.79 ± 0.46) × 10-7 M) that is statistically equal to the binding affinity in the presence of 20 nM C-peptide ((Kd = 2.05 ± 0.20) × 10-7 M). While the binding affinity between leptin and the RBC did not change with C-peptide, the moles of bound leptin did increase with C-peptide, suggesting a separate binding site on the cell for a leptin/C-peptide complex. The RBC-derived ATP increased in the presence of a leptin/C-peptide/Zn2+ addition, in a concentration-dependent manner. Control RBCs ATP release increased (71 ± 5.6%) in the presence of C-peptide and Zn2+, which increased further to (94 ± 5.6%) in the presence of Zn2+, C-peptide, and leptin.

Leptin signalling in teleost fish with emphasis in food intake regulation.

Leptin, the product of the obese (ob or Lep) gene, was first cloned in teleost fish in 2005, more than a decade after its identification in mammals. This was because bony fish and mammalian leptins share a very low amino acid sequence identity, which suggests different functionality of the leptin system in fish compared to that of mammals. Indeed, major differences are evident between the mammalian and fish leptin system. Thus, for instance, mammalian leptin is synthesized and released by the adipose tissue in response to the amount of fat depots, while several tissues (mainly the liver) are the main sources of leptin in fish, whose determining factors of production are still unclear. In mammals, the main physiological role for leptin is its involvement in the maintenance of energy balance by decreasing food intake and increasing energy expenditure, although a wide variety of actions have been attributed to this hormone (e.g., regulation of lipid and carbohydrate metabolism, reproduction and immune functions). In fish, available literature also points towards a multifunctional nature for leptin, although knowledge on its functions is limited. In this review, we offer an overview of teleostean leptin structure and mechanism of action, and discuss the available knowledge on the role of this hormone in food intake regulation in teleost fish, aiming to provide a comparative overview between the functioning of the teleostean and mammalian leptin systems.

The Central Regulation of Bone Mass: Genetic Evidence and Molecular Bases.

The alternation of resorption of preexisting bone by the osteoclasts followed by de novo bone formation by osteoblasts is called bone modeling during childhood and bone remodeling during adulthood. A central question raised by this physiological process that is fundamental to longitudinal growth during childhood and adolescence and that is attacked at the other end of life in the context of osteoporosis is to know how it is regulated. This question was rejuvenated in the late 1990s and early 2000s years when the application of mouse genetics made it feasible to test whether there were new endocrine determinants of bone (re)modeling. Addressing this question, taking into account fundamental cell biology features of bone led to the hypothesis that there should be a coordinated control of bone growth/mass, energy metabolism, and reproduction. Testing genetically and molecularly, this hypothesis revealed that, in vivo, the adipocyte-derived hormone leptin is a powerful inhibitor of bone mass accrual following its signaling in the brain. This chapter details the molecular bases and biological relevance of this regulation of bone mass accrual by leptin.

Genetic Studies of Leptin Concentrations Implicate Leptin in the Regulation of Early Adiposity.

Leptin influences food intake by informing the brain about the status of body fat stores. Rare LEP mutations associated with congenital leptin deficiency cause severe early-onset obesity that can be mitigated by administering leptin. However, the role of genetic regulation of leptin in polygenic obesity remains poorly understood. We performed an exome-based analysis in up to 57,232 individuals of diverse ancestries to identify genetic variants that influence adiposity-adjusted leptin concentrations. We identify five novel variants, including four missense variants, in LEP, ZNF800, KLHL31, and ACTL9, and one intergenic variant near KLF14. The missense variant Val94Met (rs17151919) in LEP was common in individuals of African ancestry only, and its association with lower leptin concentrations was specific to this ancestry (P = 2 × 10-16, n = 3,901). Using in vitro analyses, we show that the Met94 allele decreases leptin secretion. We also show that the Met94 allele is associated with higher BMI in young African-ancestry children but not in adults, suggesting that leptin regulates early adiposity.

An immunosensor for the determination of carcinoembryonic antigen by Surface Plasmon Resonance imaging.

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL-1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 µL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL-1 and a quantification limit of 0.40 ng mL-1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.

Cardioprotective properties of leptin in patients with excessive body mass.

BACKGROUND: Adipose tissue is producing adipokines that play different roles in the pathophysiology of cardiovascular disease. AIMS: The study aimed to assess the role of selected biomarkers in hypertensive patients with overweight and obesity compared with those with normal body-mass index (BMI). METHODS: A total of 62 patients with BMI < 25 kg/m2 (median age 54 (46-58) yrs., 57% males) and 51 with BMI ≥ 25 kg/m2 (median age 53 (48-59) yrs., 37% males) were enrolled. Biochemical parameters, leptin, adiponectin, and resistin; asymmetric dimethylarginine; interleukin 6; and N-terminal propeptide of type III procollagen, were assessed in plasma. The evaluation of hemodynamic parameters was performed using SphygmoCor 9.0 tonometer. Echocardiography was performed using AlokaAlpha 10 Premier device. RESULTS: Overweight and obese patients had significantly higher concentration of leptin (34 vs 18 ng/ml; p = 0.03), ADMA (0.43 vs 0.38 µmol/l, p = 0.04), and lower concentration of adiponectin (5.3 vs 7 µg/ml, p = 0.01). The only significant difference in tonometry analysis was higher aortic pulse pressure (mmHg) in patients with BMI ≥ 25 kg/m2 group (34 vs 30; p = 0.03). These patients had also significantly lower peak systolic velocity and early diastolic velocity in tissue Doppler imaging of the right ventricle free wall at the level of the tricuspid annulus compared with controls (p = 0.02 and p = 0.001, respectively). The level of leptin is correlated negatively with the left ventricular mass index (LVMI) (R Spearman = - 0.5; p = 0.002) and PWV (R = - 0.4; p = 0.01) and ADMA with total and LDL cholesterol (R = - 0.42; p = 0.008), and adiponectin is correlated positively with HDL cholesterol (R = 0.67; p = 0.0001). CONCLUSIONS: Leptin concentrations were inversely proportional to LVMI and PWV in patients with BMI < 25 kg/m2. TRIAL REGISTRATION: Clinicaltrials.gov study ID: NCT04175080.

The Pierced Lasso Topology Leptin has a Bolt on Dynamic Domain Composed by the Disordered Loops I and III.

Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.

Directional Immobilization of Proteins on Gold Nanoparticles Is Essential for Their Biological Activity: Leptin as a Case Study.

Gold nanomaterials hold great potential for biomedical applications. While this field is evolving rapidly, little attention has been paid to precise nanoparticle design and functionalization. Here, we show that when using proteins as targeting moieties, it is fundamental to immobilize them directionally to preserve their biological activity. Using full-length leptin as a case study, we have developed two alternative conjugation strategies for protein immobilization based on either a site-selective or a nonselective derivatization approach. We show that only nanoparticles with leptin immobilized site-selectively fully retain the ability to interact with the cognate leptin receptor. These results demonstrate the importance of a specified molecular design when preparing nanoparticles labeled with proteins.

Prediction and Structural Comparison of Deleterious Coding Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) in Human LEP Gene Associated with Obesity.

Leptin is a peptide hormone that regulates fat stores in the body and appetite by controlling the feeling of satiety. This hormone is secreted by the white adipose tissue and plays a role in the storage and mobilization of fatty acids. Mutations of the LEP gene have been associated with obesity in different populations; it is a multifactorial disease that constitutes a major public health problem. In this study, we evaluated the impact of missense SNPs in the LEP gene extracted from dbSNP using 8 computational prediction tools. Out of the total of 4337 SNPs, 93 were nsSNPs (nonsynonymous single nucleotide polymorphisms). Among 93 nsSNPs, 12 (S46L, G59S, D61N, D100N, N103K, C117S, D76V, S88C, P90R, I95N, L161R, and R105W) variants were predicted to be the most deleterious by prediction software. On these 12 deleterious SNPs, 8 variants (S46L, G59S, D61N, D100N, N103K, C117S, L161R, and R105W) were located in the conserved positions and showed a decrease in structure stability which was evaluated by I-Mutant and Mupro. Then, by analyzing the different interactions between different amino acids in wild and mutated proteins, we assessed the structural impact of the deleterious modifications using the YASARA software. Among 8 deleterious nsSNPs, we revealed structure changes in the 6 variants S46L, G59S, D100N, L103K, R105W, L161R, two of which R105W, N103K were previously reported as associated with obesity. Our study suggests 6 deleterious mutations could play an important role in contributing to human obesity and worth to be included in association and functional studies, then may be a drug target.

Breast Milk Supply of MicroRNA Associated with Leptin and Adiponectin Is Affected by Maternal Overweight/Obesity and Influences Infancy BMI.

Breast milk constitutes a dietary source of leptin, adiponectin and microRNAs (miRNAs) for newborns. Expression of miRNAs previously associated with maternal obesity, leptin or adiponectin function were assessed and their impact on infant weight analyzed. Milk samples were collected (at month 1, 2, and 3) from a cohort of 59 healthy lactating mothers (38 normal-weight and 21 overweight/obese (BMI ≥ 25)), and infant growth was followed up to 2 years of age. Thirteen miRNAs, leptin and adiponectin were determined in milk. Leptin, adiponectin and miRNA showed a decrease over time of lactation in normal-weight mothers that was altered in overweight/obesity. Furthermore, negative correlations were observed in normal-weight mothers between the expression of miRNAs in milk and the concentration of leptin or adiponectin, but were absent in overweight/obesity. Moreover, miRNAs negatively correlated with infant BMI only in normal-weight mothers (miR-103, miR-17, miR-181a, miR-222, miR-let7c and miR-146b). Interestingly, target genes of milk miRNAs differently regulated in overweight/obesity could be related to neurodevelopmental processes. In conclusion, a set of miRNAs present in breast milk, in close conjunction with leptin and adiponectin, are natural bioactive compounds with the potential to modulate infant growth and brain development, an interplay that is disturbed in the case of maternal overweight/obesity.

Leptin as a predictive marker for metabolic syndrome.

Metabolic syndrome poses a major threat on human health affecting the quality of life. Adipose tissue is an important organ which plays a crucial role in the pathogenesis of metabolic syndrome. Adipocytokines secreted by the adipose tissue plays a critical role in storage, food intake, energy expenditure, lipid and glucose metabolism. Leptin is primarily involved in regulating food intake, body weight and energy homeostasis through neuroendocrine functions. Contemporary research suggests that leptin also influences insulin sensitivity and lipid metabolism. High leptin concentrations are directly associated with the obesity subsequent development of metabolic disease sequelae such as insulin resistance, type 2 diabetes and cardiovascular diseases. Elucidation of the mechanism of action of leptin would help to develop novel therapeutic approaches for there metabolic disorders like obesity and diabetes. This review provides an updated 'state-of-the-art' about the leptin and its role in the pathophysiology of metabolic syndrome at the molecular level.

Porous graphene-black phosphorus nanocomposite modified electrode for detection of leptin.

Leptin is a vital biomarker of non-alcoholic fatty liver (NAFLD), and its evaluation of the concentration level in vivo is of great significance to NAFLD diagnosis. Therefore, it is pressing to develop a method for rapid and sensitive detection of leptin. This paper describes an environmentally friendly and label-free immunosensor based on porous graphene functionalized black phosphorus (PG-BP) composite to detect of leptin. The PG-BP was synthesized via strong coherent coupling between porous graphene (PG) surface plasmons and anisotropic black phosphorus (BP) localized surface plasmons, which made the electrochemical performance of PG and BP synergistic as well as increased the stability and conductive capability of BP material. The PG-BP modified electrodes was further prepared by gold nanoparticles, cysteamine, and glutaraldehyde in turn. Due to the cross-linking effect of glutaraldehyde, anti-leptin can be firmly fixed. These properties of the platform improved the conductive capability of the immunosensor and enhanced the load capacity of the proteins, thereby, the sensitivity of the immunosensor was significantly increased. Under the optimal conditions, the proposed immunosensor exhibited a wide linear range of 0.150-2500 pg/mL with a low detection limit of 0.036 pg/mL. The leptin immunosensor displayed excellent selectivity and anti-interference ability, which could be used for early screening and diagnosis of clinical NALFD.

Leptin Receptor as a Potential Target to Inhibit Human Testicular Seminoma Growth.

Although in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the in vivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer.

Effect of HTST and Holder Pasteurization on the Concentration of Immunoglobulins, Growth Factors, and Hormones in Donor Human Milk.

Donor human milk (DHM) is submitted to Holder pasteurization (HoP) to ensure its microbiological safety in human milk banks but this treatment affects some of its bioactive compounds. The objective of this work was to compare the effects of HoP and high temperature short time (HTST) treatments on some bioactive compounds found in DHM. A total of 24 DHM batches were processed in a continuous HTST system (70, 72, and 75°C for 5-25 s) and by HoP (62.5°C for 30 min). The concentrations of immunoglobulins (Igs) A, G, and M, transforming growth factor-beta 2 (TGF-ß2), adiponectine, ghrelin, and leptin were measured using a multiplex system, whereas the concentration of epidermal growth factor (EGF) was determined by ELISA. In relation to Igs, IgG showed the highest preservation rates (87-101%) after HTST treatments, followed by IgA (54-88%) and IgM (25-73%). Ig retention after any of the HTST treatments was higher than after HoP (p < 0.001). Treatment times required to reduce the concentration of IgM by 90% (D-value) were 130, 88, and 49 s at 70, 72, and 75°C, while the number of degrees Celsius required to change the D-value by one factor of 10 (z-value) was 11.79°C. None of the heat treatments had a significant effect on the concentrations of TGF-ß2, EGF, adiponectin, and ghrelin. In contrast, leptin was detected only in 4 of the samples submitted to HoP, whereas it was present in all samples after the different HTST treatments, with retention rates ranging between 34 and 68%. Globally, the concentration of IgA, IgG, IgM, and leptin in DHM was significantly higher after HTST pasteurization performed in a continuous system designed to be used in human milk banks than after the HoP procedure that is routinely applied at present.