Leptin-E100, the obese protein

Josephine Comas Bardot '08 and Ryan Ruffing '07

I. Introduction

Leptin, represented here as the Leptin E-100 mutant, is the product of the obesity gene (OB) discovered in 1994 by Zhang et al . Leptin is responsible for the ob/ob phenotype in obese mice. Although leptin is commonly present as its wild type, it can only be crystallized in the E-100 mutant form which substitutes glutamine for tryptophan at position 100 . This mutation has a minimal effect on the structure or function of the protein. The structure of leptin resembles the members of the long chain helical cytokine family, which are proteins that interact directly with specific cell surface receptors. Leptin, produced in adipocytes, circulates through the blood stream and provides the central nervous system (CNS) with a signal for energy intake and energy storage in the body. Leptin is more abundant when energy intake and storage are high, and less abundant when energy intake and storage are low. Thus when wild type leptin and wild type leptin receptors are present, the CNS will work with the hypothalamus to regulate appetite and maintain body weight according to the signal.

The signalling interaction between leptin and the hypothalamus is regulated by specific interactions between the protein and the leptin receptor Ob-Rb. Leptin receptors are expressed throughout the CNS, including the hypothalamus, and other body tissues. Ob-Rb and other leptin receptors that represent different splices of the same gene expressed throughout the body (Ob-Ra, Ob-Rc, Ob-Rd, Ob-Re, Ob-Rf), are part of the class I cytokine receptor family. The binding of leptin to Ob-Rb activates the signaling of transducer and activator of transcription (STAT) proteins that regulate the expression of neuronal genes affecting appetite. The hypothalamus is considered to be the main location for energy intake regulation; however, other parts of the brain can function similarly when exposed to leptin.

The continued study of leptin has focused on understanding obesity, diabetes, and cardiovascular disease in humans. Most of the research unfortunately can only use the mouse as a model which presents obstacles in understanding the function of leptin in humans. While obesity in mice can be cured by artificially increasing leptin levels in the blood stream, obese humans are resistant to similar treatments. In other words, obesity in mice is the result of mutations in the OB gene which render leptin inactive. However, obesity in humans, appears to be the result of the inability to transport leptin into CNS cells. PLEASE ACTIVATE LEPTIN RESTORE MODE

II. General Structure

The obese gene codes for a leptin molecule that is 167 amino acids in length, and includes a secretory signal sequence that is 21 amino acids in length. Thus, the protein that circulates in the body and contacts the CNS along with other body tissues is 146 amino acids long and 16 kDa in weight. This is the protein displayed here. The secondary structure of leptin is most obviously characterized by a four helix bundle which adopts an unusual up-up-down-down fold . The four helices: αA , αB , αC , and αD relate to form an antiparallel left twisted helical bundle. This left hand twisted arrangement of the four helices is antiparallel because in each of the two stacks there is an up oriented helix and a down oriented helix (A and D against B and C). Please Restore Leptin .

Four loop structures join the helices. The AB loop is an extended loop which allows helix B to run in the same direction as helix A; loop BC is short and facilitates the antiparallel BC stack; and the CD loop is long which allows C and D to both extend in the downward direction. The CD loop also includes the short and distorted helix E . Please Restore Leptin .

A large hydrophobic core of the protein runs through the center of the helix bundle and is maintained by the conserved regions of αA , αB , αC , and αD . These conserved regions relate as they face each other in the interior of the bundle . Helix E also plays an essential role in the creation of this hydrophobic core, by capping it. Please Restore Leptin .

Conserved residues from the CD loop which mostly reside in Helix E (Leu 104, Leu 107, Leu 110, Leu 114, and Val 113) , are packed closely to residues in Helix B (Val 60, Ile 64, and Met 68) and Helix D (Val 124, and Ala 125) that are hydrophobic in nature This hydrophobic cap serves to further stabilize the core of the protein as well as to hide lipophilic residues that reside on the B and D helices. Please Restore Leptin .

III. Leptin Receptors

Leptin receptors are members of the class I cytokine receptor family. Class I cytokine receptors do not have activity inside the cell therefore we say they do not have endogenous kinase activity. These receptors instead have janus kinase (JAK) activity that phosphorylates the tyrosines within Ob-Rb . Furthermore, the binding of leptin to its receptor causes the phosphorylation of the STAT protein . The phosphorylation of the STAT proteins results in the activation of transcription of the neuronal genes associated with appetite and body weight management, and completes the JAK/STAT pathway. Studies have shown that the activation of the phosphatidulinositol 3-kinase and phosphodiesterase 3B are necessary prior to the activation of the STAT proteins. The five other Ob receptors found in other cells and tissues lack the STAT protein binding, thus they are unable to activate STAT signaling. These receptors intead activate the JAK 2 and the mitogen-activated kinase, however, little is known about the physiological significance of the activation of this kinase. The Ob-Ra is better understood and it has been demonstrated that this receptor's function is to transport leptin across the blood/cerebrospinal fluid barrier in the choroid plexus. Further studies have shown that the Ob-Re receptor acts as a leptin binding protein to activate a second protein binding, which prolongs the half-life of leptin.

IV. Receptor Binding

The majority of knowledge we have about leptin receptor binding is derived from leptin's structural similarity to other long-chain helical cytokines. Though the sequences in this family of proteins differ significantly their structures are highly superimposible. Leptin receptor binding has been elucidated largely by this method of comparison because the complex of leptin and Ob-Rb has not yet been demonstrated. Within regions where other proteins of this family bind their receptors, key differences have been shown that are understood to be important in leptin binding of Ob-Rb. The first of these structural differences is the small kink in the last turn of helix D which is thought to accomodate the specific structure of leptin's receptors . Also in this same vicinity of the protein the end of helix B is about two turns shorter that in its family counterparts. The unique section at the N terminus of the D helix has also been understood as playing a key role in receptor binding . When two conserved cysteine residues, one at the N terminus of the protein and the other on the CD loop are eliminated receptor binding can not be accomplished . The disulfide bonding between these two residues is accomodated by the sharp 36 degree kink in the end of the D helix . Please Restore Leptin . Finally, there is a section of the AB loop that is distorted in the crystal structure and that remains undefined structurally . It is distorted in the structure because it is a very flexible region. This is known because similar flexible regions exist in other proteins of the family and have been stabilized in the event of receptor binding. Thus this region is believed to play a key role in the binding of Ob-Rb by leptin.

V. Leptin Induced Signaling

The study of leptin has revealed that this protein is involved in many different physiological processes besides its key role in weight homeostasis. Although many physiological pathways are controled by the CNS, the presence of leptin and its receptors throughout the entire body suggests that the protein is able to directly affect other cells and tissues. Ongoing studies have suggested that levels of leptin and proper leptin receptor expression control the possibilities for cardiovascular risk, platelet aggregation, hypertension, oxidative stress, anguigenesis, and thrombosis. Furthermore, leptin appears to have functions related to the immune system as it is present in many different immune system cells like macrophages and other bone marrow cells. Leptin works closely with insulin sensitivity to directly and indirectly affect energy intake. Just like leptin, insulin increases the activation of STAT3 and myogen-activated kinase activity explaining how leptin may influence insulin action on tissues. Other studies have shown how leptin stimulates lypolysis in white adipose tissues which results in the stimulation of the oxidation of fatty acids within the adipocytes. Although not fully understood, observations of the activity of leptin have suggested that leptin may have functions relating to reproduction. Leptin's ability to control energy intake may play a crucial role in fetal growth and development, ovarian steroidogenesis, and placental function.

V. References

Crystal Structure Information:

Zhang, Faming, et al. 1997. Crystal structure of the obese protein leptin-E100. Nature 387:206-209.

Physiological Background Information:

Prolo, Paolo, et al. 1998. Molecules in focus Leptin. The International Journal of Biochemistry & Cell Biology. 30: 1285-1290.

Considine, Robert V. 2005. Human Leptin: An Adipocyte Hormone with Weight-Regulatory and Endocrine Functions. Seminars in Vascular Medicine 5: 15-24.

Diamond, Frank B., et al. 1997. Demonstration of a Leptin Binding Factor in Human Serum. Biochemical and Biophysical research communications 233: 818-822.