A simple and rapid multiplex substrate profiling method has been developed to reveal the substrate specificity of any endo- or exo-peptidase using LC-MS/MS sequencing. A physicochemically diverse library of peptides was generated by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated prime and non-prime site information and substrate specificity matched or expanded upon previous substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C gene responsible for polypeptide processing and allowed Granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor ion abundance. Furthermore, the proteolytic secretions from a parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.Proteases are ubiquitous enzymes that function in virtually all biological phenomena. Two of the major groups of proteases, Clan CA (papain-like) cysteine proteases and Clan SA (trypsin-like) serine proteases, underwent an evolutionary inversion whereby the more abundant cysteine proteases of primitive eukaryotes gave way to serine proteases with the evolution of arthropods (1) (merops.sanger.ac.uk/). Therefore, an analysis of the role of cysteine proteases in protozoa can provide insights into differential use and molecular evolution of this protease class.Trypanosoma brucei is a protozoan parasite and the causative agent of human African trypanosomiasis, a fatal disease that is transmitted by the bite of the tsetse fly. Only four drugs are available to treat human African trypanosomiasis: two for the first stage of the disease when parasites proliferate in the blood (pentamidine and suramin) and two for the second stage when parasites have established infection in the cerebrospinal fluid (melarsoprol and eflornithine). These drugs cause serious side effects and are expensive to manufacture and administer (2). There is an obvious and urgent need to develop new chemotherapies to treat human African trypanosomiasis.Two Clan CA cysteine proteases have been identified in T. brucei: rhodesain, which is cathepsin L-like and most abundant (3), and tbcatB, a cathepsin B-like enzyme (4). Treatment of parasites in culture with the nonspecific cysteine protease inhibitor, benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-Ala-CHN 2 ) 2 is lethal to cultured parasites at 10 M (5). Parasites treated with this inhibitor exhibit altered cell morphology, are unable to undergo cytokinesis, ...

Feature matters for salient object detection. Existing methods mainly focus on designing a sophisticated structure to incorporate multi-level features and filter out cluttered features. We present Progressive Feature Polishing Network (PFPN), a simple yet effective framework to progressively polish the multi-level features to be more accurate and representative. By employing multiple Feature Polishing Modules (FPMs) in a recurrent manner, our approach is able to detect salient objects with fine details without any post-processing. A FPM parallelly updates the features of each level by directly incorporating all higher level context information. Moreover, it can keep the dimensions and hierarchical structures of the feature maps, which makes it flexible to be integrated with any CNN-based models. Empirical experiments show that our results are monotonically getting better with increasing number of FPMs. Without bells and whistles, PFPN outperforms the state-of-the-art methods significantly on five benchmark datasets under various evaluation metrics. Our code is available at: https://github.com/chenquan-cq/PFPN.

No abstract

The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1 such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k cat /K m ‫؍‬ 2 ؋ 10 5 M ؊1 s ؊1 ). Maximum hydrolysis occurs at pH 3.4 and 50°C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K i ‫؍‬ 1.5 nM), as well as a portion of the propeptide sequence, PT1 (K i ‫؍‬ 32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.As chemoheterotrophs, filamentous fungi secrete a variety of polymer-degrading hydrolases such as peptidases and carbohydrases that degrade organic material in the local environment to provide essential nutrients for the growing hyphae. Many fungi release organic acids to acidify the environment while secreting enzymes that are optimally active under these conditions. Traditionally, acid-acting fungal peptidases were assigned to the aspartic protease family and include aspergillopepsin from various Aspergillus species (1) and penicillopepsin from Penicillium species (2). These enzymes have two active-site aspartic acid residues and are strongly inhibited by pepstatin. Recently, two distinct groups of acid peptidases were identified that are insensitive to pepstatin and lack the catalytic motif observed in pepsintype peptidases. One group, "sedolisins" possess a fold similar to members of the subtilisin family but contain an active site triad of serine (S), glutamic acid (E), and aspartic acid (D) (3). They are sensitive to leupeptin and have homologs in bacteria (4), fungi (5), slime mold (6), and mammals (7). The second group of pepstatin-insensitive acid peptidases share a distinct set of structural, enzymatic, and physicochemical properties, and to date have only been identified in a select group of fungi (8). Members of this novel family possess a catalytic dyad consis...

To achieve high ionic liquid (IL) tolerance and promote in situ enzymatic hydrolysis of biomass, four metal–organic framework (MOF) carriers, including ZIF-8, UIO-66-NH2, MIL-100-Fe, and PCN-250, were used for cellulase immobilization through simple physical adsorption. The results showed that ZIF-8 had the largest enzyme adsorption capacity at 176.16 mg/g support. In addition, the immobilized cellulases activity was evaluated in the presence of 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]­DEP) by using caryboxymethyl cellulose (CMC) and filter paper (FP) as substrates. When IL concentration increased from 0% to 50% (v/v), the immobilized cellulase displayed superior IL tolerance compared with free cellulase. In particular, ZIF-8-immobilized cellulase exhibited a remarkable IL tolerance, in which the activity of caryboxymethyl cellulase (CMCase) and filter paper cellulase (FPase) were increased by 112.59% and 59.86% in 50% (v/v) [Emim]­DEP, respectively. The analysis of kinetic parameters further suggested that the immobilized cellulase had a lower equilibrium dissociation constant (k d) value and a higher final enzyme plateau activity (a p) value in a reaction system involving IL, indicating that the immobilized enzyme can effectively reduce cellulase inactivation caused by IL. Eventually, ZIF-8-immobilized cellulase with in situ hydrolysis of bagasse increased by 92.92% compared with free cellulase in 50% (v/v) [Emim]­DEP.

No abstract

No abstract