Development of analgesic peptide therapeutics for AIDS-related neuropathic pain

poster abstractChronic neuropathic pain is a huge problem to the health and well-being of an increasingly ageing population in the US, as substantiated by the large unmet clinical need associated with this type of pain, with estimates of 30-50% of sufferers refractory to existing medication. Thus, there is an imperative to increase knowledge of mechanisms of action of the key proteins in nociceptive pathways in vitro and to extend this knowledge to in vivo models of neuropathy to advance therapeutic development in this area. N-type voltage-gated Ca2+ channels (CaV2.2) have emerged as potential novel targets for the treatment of chronic neuropathic pain. Funded, in part, by a FORCES grant, we have identified two novel derivatives of the parent 15 amino acid CBD3 peptide, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to N-type voltage gated calcium channels (CaV2.2) [Brittain et al., Nature Medicine 17:822-829 (2011)]. Pharmacokinetic studies revealed nanogram levels of peptide in plasma of rats systemic administration consistent with relief of hypersensitivity. Furthermore, we observed improved and broader efficacy of the derivatized peptides in AIDS-therapy and nerve-injury related neuropathic pain models. Future studies regarding dosing and route of delivery optimization as well as identification of peptide-mimetics are ongoing to fully realize the commercial value of the peptides. Supported by the Startup program at the Indiana University Research & Technology Corporation (IURTC), we have setup Sophia Therapeutics LLC and together with IURTC are committed to the work proposed here

The Interscutularis Muscle Connectome

The complete connectional map (connectome) of a neural circuit is essential for understanding its structure and function. Such maps have only been obtained in Caenorhabditis elegans. As an attempt at solving mammalian circuits, we reconstructed the connectomes of six interscutularis muscles from adult transgenic mice expressing fluorescent proteins in all motor axons. The reconstruction revealed several organizational principles of the neuromuscular circuit. First, the connectomes demonstrate the anatomical basis of the graded tensions in the size principle. Second, they reveal a robust quantitative relationship between axonal caliber, length, and synapse number. Third, they permit a direct comparison of the same neuron on the left and right sides of the same vertebrate animal, and reveal significant structural variations among such neurons, which contrast with the stereotypy of identified neurons in invertebrates. Finally, the wiring length of axons is often longer than necessary, contrary to the widely held view that neural wiring length should be minimized. These results show that mammalian muscle function is implemented with a variety of wiring diagrams that share certain global features but differ substantially in anatomical form. This variability may arise from the dominant role of synaptic competition in establishing the final circuit.National Institutes of Health (U.S.

The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation

Autophagy is a self-degradative physiological process by which the cell removes worn-out or damaged components. Constant at basal level it may become highly active in response to cellular stress. The type 2 transglutaminase (TG2), which accumulates under stressful cell conditions, plays an important role in the regulation of autophagy and cells lacking this enzyme display impaired autophagy/mitophagy and a consequent shift their metabolism to glycolysis. To further define the molecular partners of TG2 involved in these cellular processes, we analysed the TG2 interactome under normal and starved conditions discovering that TG2 interacts with various proteins belonging to different functional categories. Herein we show that TG2 interacts with pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis which is responsible for maintaining a glycolytic phenotype in malignant cells and displays non metabolic functions, including transcriptional co-activation and protein kinase activity. Interestingly, the ablation of PKM2 led to the decrease of intracellular TG2's transamidating activity paralleled by an increase of its tyrosine phosphorylation. Along with this, a significant decrease of ULK1 and Beclin1 was also recorded, thus suggesting a block in the upstream regulation of autophagosome formation. These data suggest that the PKM2/TG2 interplay plays an important role in the regulation of autophagy in particular under cellular stressful conditions such as those displayed by cancer cells

The Significance of the J-Curve in Hypertension and Coronary Artery Diseases

The J-curve effect describes an inverse relation between low blood pressure (BP) and cardiovascular complications. This effect is more pronounced in patients with preexisting coronary artery disease (CAD), hypertension or left ventricular hypertrophy (LVH). The recent large clinical outcomes trials have observed a J-curve effect between a diastolic BP of 70-80 mmHg as well as a systolic BP <130 mmHg. The J-curve phenomenon does not appear in stroke or renal disease. This is because the coronary arteries are perfused during diastole, but the cerebral and renal perfusion mainly occurs in systole. Therefore, caution should be taken to maintain the diastolic blood pressure (DBP) at minimum of 70 mmHg and possibly to maintain the DBP between 80-85 mmHg in patients with severe LVH, CAD or vascular diseases. BP control in high-risk elderly patients should be carefully done as undergoing aggressive therapy to lower the systolic blood pressure below 140 mmHg can cause cardiovascular complications due to the severely reduced DBP and increased pulse pressure

Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data