Glucagon-like Peptide-1

Glucagon-like Peptide-1 (GLP-1) is one of the members of incretin, a group of gastrointestinal hormones secreted by endocrine cells in small intestine that mainly work to increase insulin levels. Under normal physiological condition, this 30-amino acid peptide is secreted by L cells, a kind of neuroendocrine cell located in small intestine after a meal, into the circulation, promoting insulin release and inhibit energy intake. Its natural form can be degraded by DPP-4, having a half life of 1-2 minutes. GLP-1 mainly acts on the GLP-1 receptor, a G-protein coupled receptors, coupled with adenylyl cyclase downstream and signaling on PI3K/Akt and ERK1/2 pathways via second messenger cAMP. It promotes the secretion of insulin in β cells of islets in pancreas, antagonizes and glucose-rising effect of glucagon, and largely increase the insulin sensitivity of targeted cells. GLP-1 analogues such as Liraglutide and Exentide and DPP-4 inhibitors have been used clinically as treatment for patients with type 2 diabetes mellitus (T2DM), but recently, more and more focus has been spotted on its versatile functions, such as cardiovascular regulation, appetite inhibition, and neuroprotective effects. Here we aim to utilize the functions in appetite inhibition and neuroprotection of GLP-1, in order to tailor our PEPDEX as a living machine that communicate with our central nervous systems and modulate our eating behavior, as well as improve our cognitive function to prevent neurodegenerative diseases.

Versatile function of GLP-1

Fat Extinguisher—Reduction in Eating Behavior

Obesity and metabolic syndrome have been regarded as chronic disease recent decades. It is reported that people with obesity or metabolic syndromes are in a higher risk of type II diabetes, cardiovascular disease and hypertension. Nowadays, there are 1 billion people encountering over-weight problem, and 30 million people are diagnosed of obesity. Modern dietary pattern has been considered the major cause contributing to the phenomena. GLP-1 is known to act on the satiety center inside hypothalamic regions of brains, inhibiting appetite and modulating feeding behavior. Therapeutic use of anti-obesity has also been proved by different drug authorities. In PepdEx, we put GLP-1 production under the control of a circuit with a fatty acid sensor, mimicking the physiological response that GLP-1 is released after food intake. Induced by a fat meal, bacterial secreted GLP-1 are expected to penetrate the intestinal epithelium with the assistance of cell penetrating peptide Penetratin, circulating to the brain and perform its behavior-regulating function.

Brain Guardian—Memory Improvement and Neuroprotection

Besides regulation of appetite, GLP-1 also has wide effects on the central nervous system, including promotion of learning and memory and neuroprotective roles. GLP-1 receptors are distributed among the brain in various regions such as hypothalamus, thalamus, brainstem, and hippocampus. Studies using in vitro culture and animal models indicate that GLP-1 promote the learning and memory function in CA1 region of hippocampus, via increase of long-term potentiation (LTP) and enhancement of synaptic plasticity. It also reduces the destructive effects of Aβ amyloid and preserve and dopaminergic neurons in Alzheimer’s disease and Parkinson’s disease, respectively. Potentiation of neuronal differentiation, promotion of cell survival, up-regulation of neurotransmitter synthesis and modulation of glial cells are other functions of GLP-1 in CNS that have been reported, while more diseases such as Huntington’s disease, stroke, and peripheral sensory neuropathy potentially become its therapeutic targets. Conjugation with a thermal regulatory promoter gives a constitutive delivery of GLP-1 inside the human body, which constantly stimulates and protects the CNS, in improvement of learning and memory and prevention of neurodegenerative diseases.

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Cell Penetrating Peptide

One obstacle needs to be overcome is the poor penetrating ability of GLP-1 through the intestinal epithelia, since GLP-1 is neither small molecule nor hydrophobic. However, several studies have shown that some short peptides known as cell penetrating peptides (CPPs) can help to deliver biodrugs through intestinal mucosa and increase their bioavailability. CPPs are usually arginine-rich, tend to interact with extracellular matrix and can enhance macropinocytosis of epithelial cells. One recent study even demonstrated that co-administration of GLP-1 and Penetratin, one of the CPPs, greatly increased the intestinal absorption rate to 5%. By applying cell penetrating peptide to our device, we aim to promote the delivery of GLP-1 from intestinal lumen to blood stream, therefore enhance their appetite-regulating effect.

Transepithelial effect of CPP

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Secretion

In order to achieve the extracellular secretion of our recombinant peptide GLP-1 and CPP, we utilize the endogenous type 2 secretion system (T2SS) in bacteria with specific signal peptides added to the N-terminus of GLP-1 and CPP.

Type 2 Secretion System

Secretion systems of gram-negative bacteria are categorized into 6 types, from type 1 secretion system (T1SS) to type 6 secretion system (T6SS). Among these secretion systems, type 1 and type 2 secretion systems are in charge of transport bacterial protein products into the extracellular space. [1] Here we choose type 2 secretion system (T2SS) as our secretion apparatus which is widely found in gram-negative bacteria, including E. coli. Unlike the single-step secretion of T1SS such as hemolysin transport system (Hly), T2SS has two-step secretion processes. Unfolded or folded proteins with specific signal sequences on their N-terminus are first transported through the inner membrane into the periplasmic space via Sec translocon or twin-arginine translocation (Tat) pathway, respectively. [1, 4, 5] The cleavage of signal peptides is performed by signal peptidase inside the periplasm. Proteins of proper structural features are then transported through the outer membrane into the extracellular space via general secretory pathway (GSP) which consists of 4 distinguished subassemblies: inner membrane platform, outer membrane complex, pseudopilus, and secretion ATPase (Fig. 1) [2, 3] , while small proteins (in our case, short peptides) can also leak from the periplasmic into the environment, regarding the integrity of the outer membrane. Co-expressions of some genes, such as kil, out, tolAIII, and bacteriocin release protein (BRP), were reported to facilitate the extracellular secretory processes. [1]

Summary of Type 2 Secretion System

Signal Peptides

The selection of signal sequence for delivery of specific recombinant proteins is mostly based on empirical data. Some representative signal sequences, such as those of PhoA (alkaline phosphatase), PelB (pectate lysase B), OmpA (outer-membrane protein A), OmpF (outer-membrane protein F), StII (heat-stable enterotoxin 2), or LTB (heat-labile enterotoxin subunit B), are commonly used. We survey papers of successfully secretion of recombinant proteins with specific signal sequences, and choose 3 sequences which were reported to work in the bacterial strains (E. coli DH5α and BL21) we use. They are signal sequences of PhoA [6] , LTB [7] , and StII [8] , named signal peptide 1 (SP1), signal peptide 2 (SP2), and signal peptide 3 (SP3), respectively. Targeting to the Sec pathway, these signal sequences are cleaved in the periplasmic space, which helps to preserve the structural and functional integrity of the original the peptide. This is also an advantage to choose the T2SS as our delivery system.

Reference

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