Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than fulllength huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeatcontaining huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.
Etanercept (Amgen Inc, Thousand Oaks, CA) is a human soluble p75 tumor necrosis factor (TNF) receptor-human-IgG1 (hup75 TNFR-huIgG1) fusion protein used in the treatment of chronic inflammatory diseases in humans, including rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis. To be able to study the effects of the soluble receptor fusion protein in mouse models, including those that mimic human granulomatous infections, a murine soluble p75-TNF receptor-murine IgG1 (murine p75-murine IgG1) fusion protein had to be constructed. This article discusses the generation, large-scale production, and purification of this molecule.
Background and Purpose: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure.Experimental Approach: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain.Key Results: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin.The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats.In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric Abbreviations: albVHH, albumin binding variable heavy chain domain of a heavy-chain antibody; CMC, chemistry, manufacturing, and controls; FcRn, neonatal Fc receptor; NHP, nonhuman primate; RXFP1, relaxin family peptide receptor 1; RXFP2, relaxin family peptide receptor 2; SPR, surface plasmon resonance.
The tripeptide Lys-Cys-Lys has been synthesized and covalently labeled at the cysteine sulfhydryl with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to produce a fluorescent labeled peptide (FLP). When excited at 340 nm, the FLP fluorescence strongly with maximal intensity at 405 nm. Addition of proteins containing the kringle lysine-binding domain, such as human lipoprotein (a) and plasminogen kringle 4, significantly attenuate the fluorescence intensity of the FLP. Other proteins, such as bovine serum albumin, did not affect the quantum yield of FLP fluorescence. When human lipoprotein (a) is bound to a lysine-Sepharose affinity column, FLP was found to effectively elute the protein, indicating that the peptide can compete with lysine for the kringle-binding site on lipoprotein (a). The data suggest that FLP binds specifically to kringles through the lysine residues on the peptide, and that binding significantly affects the fluorescence from the labeled peptide. These properties of FLP make it a potentially useful tool for studying the relative affinity of different kringles for lysine binding, which is thought to be an important mechanism for kringle-target protein interactions.
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