Origin and expansion of the serine protease repertoire in the myelomonocyte lineage.

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold

In vitro and in vivo evaluation of doxorubicin conjugates with the divalent peptide E-[c(RGDfK)2] that targets integrin alphavbeta3

Integrins, especially integrin alpha vbeta 3, are attractive receptors for vascular targeting strategies. Recently, a divalent RGD peptidomimetic, E-[c(RGDfK) 2], has been described that demonstrates increased uptake in human ovarian carcinoma OVCAR-3 xenograft tumors. Inspired by these results, we set out to develop doxorubicin conjugates with E-[c(RGDfK) 2] by binding two different maleimide derivatives of doxorubicin to E-[c(RGDfK) 2] that was thiolated with iminothiolane. In this way, two water-soluble derivatives were obtained, E-[c(RGDfK) 2]-DOXO-1 and E-[c(RGDfK) 2]-DOXO-2. In E-[c(RGDfK) 2]-DOXO-1, doxorubicin was bound to the peptide through a stable amide bond, and in E-[c(RGDfK) 2]-DOXO-2, a MMP-2/MMP-9 cleavable octapeptide was introduced between doxorubicin and the peptide. The rationale for a MMP-2/MMP-9-cleavable linker was that MMP-2 and MMP-9 bind to integrin alpha vbeta 3 and both are overexpressed in tumor vasculature. In addition, analogous control doxorubicin-containing peptides bearing c(RADfK) that does not bind to integrin alpha vbeta 3 were synthesized, i.e., c(RADfK)-DOXO-1 and c(RADfK)-DOXO-2. Whereas E-[c(RGDfK) 2]-DOXO-2 was cleaved effectively by MMP-2 and in OVCAR-3 tumor homogenates releasing a doxorubicin-tetrapeptide or doxorubicin as the final cleavage product, no release of doxorubicin was observed for E-[c(RGDfK) 2]-DOXO-1. Proliferation of HUVEC in the presence of MMP-2-cleavable doxorubicin-containing peptides exhibited 6- to 10-fold increased inhibition compared to the amide-linked doxorubicin-containing peptides. In addition, inhibition of HUVEC sprouting during a 24 h exposure was approximately 3-fold stronger for E-[c(RGDfK) 2]-DOXO-2 and 20-fold stronger for the reference peptide conjugate c(RADfK)-DOXO-2 than for doxorubicin alone. In vivo studies in an OVCAR-3 xenograft model demonstrated no or only moderate antitumor efficacy for either E-[c(RGDfK) 2], E-[c(RGDfK) 2]-DOXO-1, E-[c(RGDfK) 2]-DOXO-2, or c(RADfK)-DOXO-2, even at doses of 3 x 24 mg/kg doxorubicin equivalents, compared to an improved antitumor effect for doxorubicin at 2 x 8 mg/kg

The yeast ER-intramembrane protease Ypf1 refines nutrient sensing by regulating transporter abundance.

Proteolysis by aspartyl intramembrane proteases such as presenilin and signal peptide peptidase (SPP) underlies many cellular processes in health and disease. Saccharomyces cerevisiae encodes a homolog that we named yeast presenilin fold 1 (Ypf1), which we verify to be an SPP-type protease that localizes to the endoplasmic reticulum (ER). Our work shows that Ypf1 functionally interacts with the ER-associated degradation (ERAD) factors Dfm1 and Doa10 to regulate the abundance of nutrient transporters by degradation. We demonstrate how this noncanonical branch of the ERAD pathway, which we termed "ERAD regulatory" (ERAD-R), responds to ligand-mediated sensing as a trigger. More generally, we show that Ypf1-mediated posttranslational regulation of plasma membrane transporters is indispensible for early sensing and adaptation to nutrient depletion. The combination of systematic analysis alongside mechanistic details uncovers a broad role of intramembrane proteolysis in regulating secretome dynamics

Role of kallikrein 7 in body weight and fat mass regulation.

Increased plasma and adipose tissue protease activity is observed in patients with type 2 diabetes and obesity. It has been proposed that specific proteases contribute to the link between obesity, adipose tissue inflammation and metabolic diseases. We have recently shown that ablation of the serine protease kallikrein-related peptidase 7 (Klk7) specifically in adipose tissue preserves systemic insulin sensitivity and protects mice from obesity-related AT inflammation. Here, we investigated whether whole body Klk7 knockout (Klk7-/-) mice develop a phenotype distinct from that caused by reduced Klk7 expression in adipose tissue. Compared to littermate controls, Klk7-/- mice gain less body weight and fat mass both under chow and high fat diet (HFD) feeding, are hyper-responsive to exogenous insulin and exhibit preserved adipose tissue function due to adipocyte hyperplasia and lower inflammation. Klk7-/- mice exhibit increased adipose tissue thermogenesis, which is not related to altered thyroid function. These data strengthen our recently proposed role of Klk7 in the regulation of body weight, energy metabolism, and obesity-associated adipose tissue dysfunction. The protective effects of Klk7 deficiency in obesity are likely linked to a significant limitation of adipocyte hypertrophy. In conclusion, our data indicate potential application of specific KLK7 inhibitors to regulate KLK7 activity in the development of obesity and counteract obesity-associated inflammation and metabolic diseases

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance