Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech.

When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl3-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.

An undefined cystatin CsCPI1 from tea plant Camellia sinensis harbors antithrombotic activity.

Tea consumption has been linked to a decreased risk of cardiovascular disease (CVD) mortality, which imposes a heavy burden on the healthcare system; however, which components in tea cause this beneficial effect is not fully understood. Here we uncovered a cystatin (namely CsCPI1), which is a cysteine proteinase inhibitor (CPI) of the tea plant (Camellia sinensis) that promotes antithrombotic activity. Since thrombosis is a common pathogenesis of fatal CVDs, we investigated the effects of CsCPI1, which showed good therapeutic effects in mouse models of thrombotic disease and ischemic stroke. CsCPI1 significantly increases endothelial cell production of nitric oxide (NO) and inhibits platelet aggregation. Notably, CsCPI1 exhibited no cytotoxicity or resistance to pH and temperature changes, which indicates that CsCPI1 might be a potent antithrombotic agent that contributes to the therapeutic effects of tea consumption against CVD. Specifically, the antithrombotic effects of CsCPI1 are distinct from the classical function of plant cystatins against herbivorous insects. Therefore, our study proposes a new potential role of cystatins in CVD prevention and treatment, which requires further study.

Targeting surface nucleolin induces autophagy-dependent cell death in pancreatic cancer via AMPK activation.

Pancreatic cancer remains one of the deadliest human cancers despite current advances in conventional therapeutics including surgery and adjuvant therapies. Here, we showed that LZ1, a peptide derived from a snake venom cathelicidin, significantly inhibited growth of pancreatic cancer cells by inducing autophagy-dependent cell death both in vitro and in vivo. The LZ1-induced cell death was blocked by pharmacological or genetic inhibition of autophagy. In orthotopic model of pancreatic cancer, systemic administration of LZ1 (1-4 mg/kg) exhibited remarkable antitumor efficacy, significantly prolonged mice survival, and showed negligible adverse effects by comparison with gemcitabine (20 mg/kg). Mechanistic studies revealed that LZ1 acts through binding to nucleolin, whose expression on cell surface is frequently increased in pancreatic cancer cells. LZ1 binding triggers degradation of surface-expressed nucleolin. This leads to activation of 5'-AMP kinase which results in suppression of mTORC1 activity and induction of autophagic flux. These data suggest that LZ1, targeting nucleolin-AMPK-autophagy axis, is a promising lead for the development of therapeutic agents against pancreatic cancer.

Isolation and Characterization of Poecistasin, an Anti-Thrombotic Antistasin-Type Serine Protease Inhibitor from Leech Poecilobdella manillensis.

Antistasin, first identified as a potent inhibitor of the blood coagulation factor Xa, is a novel family of serine protease inhibitors. In this study, we purified a novel antistasin-type inhibitor from leech Poecilobdella manillensis called poecistasin. Amino acid sequencing of this 48-amino-acid protein revealed that poecistasin was an antistasin-type inhibitor known to consist of only one domain. Poecistasin inhibited factor XIIa, kallikrein, trypsin, and elastase, but had no inhibitory effect on factor Xa and thrombin. Poecistasin showed anticoagulant activities. It prolonged the activated partial thromboplastin time and inhibited FeCl3-induced carotid artery thrombus formation, implying its potent function in helping Poecilobdella manillensis to take a blood meal from the host by inhibiting coagulation. Poecistasin also suppressed ischemic stroke symptoms in transient middle cerebral artery occlusion mice model. Our results suggest that poecistasin from the leech Poecilobdella manillensis plays a crucial role in blood-sucking and may be an excellent candidate for the development of clinical anti-thrombosis and anti-ischemic stroke medicines.

Antimicrobial peptide LL-37 forms complex with bacterial DNA to facilitate blood translocation of bacterial DNA and aggravate ulcerative colitis.

Bacterial DNA (bacDNA) is frequently found in serum of patient with ulcerative colitis (UC) and Crohn's disease, even blood bacterial culture is negative. How bacDNA evades immune elimination and is translocated into blood remain unclear. Here, we showed that bacDNA avoids elimination and disables bacteria-killing function of antimicrobial peptide LL-37 (Cramp in mice) by forming complex with LL-37, which is inducible after culture with bacteria or bacterial products. Elevated LL-37-bacDNA complex was found in plasma and lesions of patients with UC. LL-37-bacDNA promoted inflammation by inducing Th1, Th2 and Th17 differentiation and activating toll-like receptor-9 (TLR9). The complex also increased paracellular permeability, which possibly combines its inflammatory effects to promote local damage and bacDNA translocation into blood. Cramp-bacDNA aggravated mouse colitis severity while interference with the complex ameliorated the disease. The study identifies that inflammatogenic bacDNA utilizes LL-37 as a vehicle for blood translocation and to evade immune elimination. Additionally, bacteria may make a milieu by releasing bacDNA to utilize and resist host antimicrobial peptides as a 'trojan horse'.

An insecticidal toxin from Nephila clavata spider venom.

Spiders are the most successful insect predators given that they use their venom containing insecticidal peptides as biochemical weapons for preying. Due to the high specificity and potency of peptidic toxins, discoveries of insecticidal toxins from spider venom have provided an opportunity to obtain natural compounds for agricultural applications without affecting human health. In this study, a novel insecticidal toxin (µ-NPTX-Nc1a) was identified and characterized from the venom of Nephila clavata. Its primary sequence is GCNPDCTGIQCGWPRCPGGQNPVMDKCVSCCPFCPPKSAQG which was determined by automated Edman degradation, cDNA cloning, and MS/MS analysis. BLAST search indicated that Nc1a shows no similarity with known peptides or proteins, indicating that Nc1a belongs to a novel family of insecticidal peptide. Nc1a displayed inhibitory effects on NaV and KV channels in cockroach dorsal unpaired median neurons. The median lethal dose (LD50) of Nc1a on cockroach was 573 ng/g. Herein, a study that identifies a novel insecticidal toxin, which can be a potential candidate and/or template for the development of bioinsecticides, is presented.

Purification and function of two analgesic and anti-inflammatory peptides from coelomic fluid of the earthworm, Eisenia foetida.

The potential application of anti-inflammatory and analgesic compounds in medication and therapeutic care have become of increasing interest. We purified and characterized two novel analgesic and anti-inflammatory peptides, VQ-5 and AQ-5, from the coelomic fluid of the earthworm (Eisenia foetida). Their primary structures were determined as VSSVQ and AMADQ, respectively. Both peptides, especially AQ-5, exhibited analgesic activity in mouse models of persistent neuropathic pain and inflammation. AQ-5 also inhibited tumor necrosis factor alpha and cyclooxygenase-2 production. The mitogen-activated protein kinase signaling pathway, which is involved in analgesic and anti-inflammatory functions, was inhibited by AQ-5. Thus, the analgesic and anti-inflammatory effects of these peptides, especially AQ-5, demonstrated their potential as candidates for the development of novel analgesic medicines.