Distinct Substrate Preferences Research Articles

Functional Characterization of Long-Chain Acyl-CoA Synthetase Gene Family from the Oleaginous Alga Chromochloris zofingiensis.

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid β-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.

Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase.

Many bacteria produce and use extracellular signaling molecules such as acyl homoserine lactones (AHLs) to communicate and coordinate behavior in a cell-density dependent manner, via a communication system called quorum sensing (QS). This system regulates behaviors including but not limited to virulence and biofilm formation. We focused on Pseudomonas aeruginosa, a human opportunistic pathogen that is involved in acute and chronic lung infections and which disproportionately affects people with cystic fibrosis. P. aeruginosa infections are becoming increasingly challenging to treat with the spread of antibiotic resistance. Therefore, QS disruption approaches, known as quorum quenching, are appealing due to their potential to control the virulence of resistant strains. Interestingly, P. aeruginosa is known to simultaneously utilize two main QS circuits, one based on C4-AHL, the other with 3-oxo-C12-AHL. Here, we evaluated the effects of signal disruption on 39 cystic fibrosis clinical isolates of P. aeruginosa, including drug resistant strains. We used two enzymes capable of degrading AHLs, known as lactonases, with distinct substrate preference: one degrading 3-oxo-C12-AHL, the other degrading both C4-AHL and 3-oxo-C12-AHL. Two lactonases were used to determine the effects of signal disruption on the clinical isolates, and to evaluate the importance of the QS circuits by measuring effects on virulence factors (elastase, protease, and pyocyanin) and biofilm formation. Signal disruption results in at least one of these factors being inhibited for most isolates (92%). Virulence factor activity or production were inhibited by up to 100% and biofilm was inhibited by an average of 2.3 fold. Remarkably, the treatments led to distinct inhibition profiles of the isolates; the treatment with the lactonase degrading both signaling molecules resulted in a higher fraction of inhibited isolates (77% vs. 67%), and the simultaneous inhibition of more virulence factors per strain (2 vs. 1.5). This finding suggests that the lactonase AHL preference is key to its inhibitory spectrum and is an essential parameter to improve quorum quenching strategies.

Determination of the crystal structure and substrate specificity of ananain

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M−1s−1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E−64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E−64 and ananain explains the high inhibitory efficiency of E−64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

Phylogenetic analysis reveals key residues in substrate hydrolysis in the isomaltase domain of sucrase-isomaltase and its role in starch digestion

BackgroundStarch constitutes one of the main sources of nutrition in the human diet and is broken down through a number of stages of digestion. Small intestinal breakdown of starch-derived substrates occurs through the mechanisms of small intestinal brush border enzymes, maltase-glucoamylase and sucrase-isomaltase. These enzymes each contain two functional enzymatic domains, and though they share sequence and structural similarities due to their evolutionary conservation, they demonstrate distinct substrate preferences and catalytic efficiency. The N-terminal isomaltase domain of sucrase-isomaltase has a unique ability to actively hydrolyze isomaltose substrates in contrast to the sucrase, maltase and glucoamylase enzymes. MethodsThrough phylogenetic analysis, structural comparisons and mutagenesis, we were able to identify specific residues that play a role in the distinct substrate preference. Mutational analysis and comparison with wild-type activity provide evidence that this role is mediated in part by affecting interactions between the sucrase and isomaltase domains in the intact molecule. ResultsThe sequence analysis revealed three residues proposed to play key roles in isomaltase specificity. Mutational analysis provided evidence that these residues in isomaltase can also affect activity in the partner sucrase domain, suggesting a close interaction between the domains. Major conclusionsThe sucrase and isomaltase domains are closely interacting in the mature protein. The activity of each is affected by the presence of the other.General Significance: There has been little experimental evidence previously of the effects on activity of interactions between the sucrase-isomaltase enzyme domains. By extension, similar interactions might be expected in the other intestinal α-glucosidase, maltase-glucoamylase.

Linking bioenergetic function of mitochondria to tissue-specific molecular fingerprints.

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.

Two new taxa of the Auriscalpium vulgare species complex with substrate preferences

Basidiomes of the cone-inhabiting Auriscalpium species, usually regarded as A. vulgare Gray, are widely distributed across the Northern Hemisphere and easily recognized because of their unique macro-morphology. However, phylogenetic diversity patterns among different geographic populations and different substrates are completely unknown. In this study, samples of A. vulgare s.l. in different areas of China were studied phylogenetically, morphologically, and ecologically. For comparison, European collections of A. vulgare were included. Our phylogenetic analyses, inferred from partial nucleotide sequences including the internal transcribed spacers (ITS) 1 and 2 with the 5.8S nrDNA, the large subunit of nuclear ribosomal RNA (nrLSU), and the region between the conserved domain 6 and 7 of the gene for the second largest subunit of RNA polymerase II (RPB2), indicated that three phylogenetic species can be recognized, which correspond well with morphological and ecological evidence. Therefore, three species, having distinct substrate preferences, are documented here, namely the well-known A. vulgare, and the newly described species A. orientale and A. microsporum. Auriscalpium vulgare is widely distributed throughout the northern temperate Eurasia and North America, while the new species have been known mainly from subtropical East Asia and northeastern India to date.

NAT/NCS2-hound: a webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase-cation symporters of the NAT/NCS2 family.

Nucleobase transporters are important for supplying the cell with purines and/or pyrimidines, for controlling the intracellular pool of nucleotides, and for obtaining exogenous nitrogen/carbon sources for metabolism. Nucleobase transporters are also evaluated as potential targets for antimicrobial therapies, since several pathogenic microorganisms rely on purine/pyrimidine salvage from their hosts. The majority of known nucleobase transporters belong to the evolutionarily conserved and ubiquitous nucleobase-ascorbate transporter/nucleobase-cation symporter-2 (NAT/NCS2) protein family. Based on a large-scale phylogenetic analysis that we performed on thousands of prokaryotic proteomes, we developed a webserver that can detect and distinguish this family of transporters from other homologous families that recognize different substrates. We can further categorize these transporters to certain evolutionary groups with distinct substrate preferences. The webserver scans whole proteomes and graphically displays which proteins are identified as NAT/NCS2, to which evolutionary groups and subgroups they belong to, and which conserved motifs they have. For key subgroups and motifs, the server displays annotated information from published crystal-structures and mutational studies pointing to key functional amino acids that may help experts assess the transport capability of the target sequences. The server is 100% accurate in detecting NAT/NCS2 family members. We also used the server to analyze 9,109 prokaryotic proteomes and identified Clostridia, Bacilli, β- and γ-Proteobacteria, Actinobacteria, and Fusobacteria as the taxa with the largest number of NAT/NCS2 transporters per proteome. An analysis of 120 representative eukaryotic proteomes also demonstrates the server's capability of correctly analyzing this major lineage, with plants emerging as the group with the highest number of NAT/NCS2 members per proteome.

\u03b2-Glucuronidases of opportunistic bacteria are the major contributors to xenobiotic-induced toxicity in the gut

Gut bacterial β-D-glucuronidases (GUSs) catalyze the removal of glucuronic acid from liver-produced β-D-glucuronides. These reactions can have deleterious consequences when they reverse xenobiotic metabolism. The human gut contains hundreds of GUSs of variable sequences and structures. To understand how any particular bacterial GUS(s) contributes to global GUS activity and affects human health, the individual substrate preference(s) must be known. Herein, we report that representative GUSs vary in their ability to produce various xenobiotics from their respective glucuronides. To attempt to explain the distinct substrate preference, we solved the structure of a bacterial GUS complexed with coumarin-3-β-D-glucuronide. Comparisons of this structure with other GUS structures identified differences in loop 3 (or the α2-helix loop) and loop 5 at the aglycone-binding site, where differences in their conformations, hydrophobicities and flexibilities appear to underlie the distinct substrate preference(s) of the GUSs. Additional sequence, structural and functional analysis indicated that several groups of functionally related gut bacterial GUSs exist. Our results pinpoint opportunistic gut bacterial GUSs as those that cause xenobiotic-induced toxicity. We propose a structure-activity relationship that should allow both the prediction of the functional roles of GUSs and the design of selective inhibitors.

How ERAP1 and ERAP2 Shape the Peptidomes of Disease-Associated MHC-I Proteins.

Four inflammatory diseases are strongly associated with Major Histocompatibility Complex class I (MHC-I) molecules: birdshot chorioretinopathy (HLA-A*29:02), ankylosing spondylitis (HLA-B*27), Behçet's disease (HLA-B*51), and psoriasis (HLA-C*06:02). The endoplasmic reticulum aminopeptidases (ERAP) 1 and 2 are also risk factors for these diseases. Since both enzymes are involved in the final processing steps of MHC-I ligands it is reasonable to assume that MHC-I-bound peptides play a significant pathogenetic role. This review will mainly focus on recent studies concerning the effects of ERAP1 and ERAP2 polymorphism and expression on shaping the peptidome of disease-associated MHC-I molecules in live cells. These studies will be discussed in the context of the distinct mechanisms and substrate preferences of both enzymes, their different patterns of genetic association with various diseases, the role of polymorphisms determining changes in enzymatic activity or expression levels, and the distinct peptidomes of disease-associated MHC-I allotypes. ERAP1 and ERAP2 polymorphism and expression induce significant changes in multiple MHC-I-bound peptidomes. These changes are MHC allotype-specific and, without excluding a degree of functional inter-dependence between both enzymes, reflect largely separate roles in their processing of MHC-I ligands. The studies reviewed here provide a molecular basis for the distinct patterns of genetic association of ERAP1 and ERAP2 with disease and for the pathogenetic role of peptides. The allotype-dependent alterations induced on distinct peptidomes may explain that the joint association of both enzymes and unrelated MHC-I alleles influence different pathological outcomes.

The benefits and risks of expressing the POT and FOT family of oligopeptide transporters in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, all strains possess a gene for the evolutionarily conserved POT family peptide transporter, Ptr2; however, the genes for a novel FOT family transporter were found only in some wine brewing strains. The substrate specificity of the POT and FOT family of transporters was compared. Among the naturally occurring oligopeptides that were tested, Lys-Leu and Arg-Phe were Ptr2-specific substrates. Artificial dipeptide aspartame was imported specifically through the FOT transporter, but the structurally similar Asp-Phe was a substrate of both FOT and Ptr2 transporters. Furthermore, only the FOT transporter was important for high sensitivity to an antibiotic puromycin. These results demonstrate that the POT and FOT family of transporters have distinct substrate preferences although both transporters import overlapping dipeptide substrates. Having POT and FOT transporters is advantageous for cells to acquire nutrients, but also detrimental when these cells are exposed to the toxic molecules of their substrates.

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex.

Grc3 is an essential well-conserved eukaryotic polynucleotide kinase (PNK) that cooperates with the endoribonuclease Las1 to process the preribosomal RNA (rRNA). Aside from being dependent upon Las1 for coordinated kinase and nuclease function, little is known about Grc3 substrate specificity and the molecular mechanisms governing kinase activity. Here we characterize the kinase activity of Grc3 and identify key similarities and differences between Grc3 and other polynucleotide kinase family members. In contrast to other PNK family members, Grc3 has distinct substrate preference for RNA substrates in vitro. By disrupting conserved residues found at the Grc3 kinase active site, we identified specific residues required to support Grc3-directed Las1-mediated pre-rRNA cleavage in vitro and in vivo. The crosstalk between Grc3 and Las1 ensures the direct coupling of cleavage and phosphorylation during pre-rRNA processing. Taken together, our studies provide key insight into the polynucleotide kinase activity of the essential enzyme Grc3 and its molecular crosstalk with the endoribonuclease Las1.

Two novel deep-sea sediment metagenome-derived esterases: residue 199 is the determinant of substrate specificity and preference

BackgroundThe deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods.ResultsTwo novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35–40 °C and were found to be resistant to some metal ions (Ba2+, Mg2+, and Sr2+) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2–C8, whereas DMWf18-558 presented a wider range of C2–C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543.ConclusionsThis study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.

Characterization of the deubiquitination activity and substrate specificity of the chicken ubiquitin-specific protease 1/USP associated factor 1 complex.

Deubiquitinases (DUBs) are essential regulators of intracellular processes involving ubiquitin (Ub) modification. The human DUB ubiquitin-specific protease 1 (hUSP1) interacts with human USP-associated factor 1 (hUAF1), and helps to regulate processes such as DNA damage repair. Previously, we identified a chicken USP1 homologue (chUSP1) during an investigation into the properties of Marek's disease virus (MDV). However, chUSP1's deubiquitination activity, interaction with chUAF1, and substrate specificity remained unknown. In the present study, we expressed and purified both chUAF1 and chUSP1 with or without putative catalytic core mutations using the Bac-to-Bac system, before investigating their deubiquitination activity and kinetics using various substrates. chUSP1 was shown to interact with chUAF1 both in cellular assays in which the two proteins were co-expressed, and in in vitro assays using purified proteins. Heterodimerization with chUAF1 increased the deubiquitination activity of chUSP1 up to 54-fold compared with chUSP1 alone. The chUSP1 mutants C91S, H603A, and D758A reduced the deubiquitination activity of the chUSP1/chUAF1 complex by 10-, 7-, and 33-fold, respectively, while the C91A and H594A chUSP1 mutants eliminated deubiquitination activity of the chUSP1/chUAF1 complex completely. This suggests that C91 and H594, but not D758, are essential for chUSP1 deubiquitination activity, and that a nucleophilic group at position 91 is needed for the deubiquitination reaction. The chUSP1/chUAF1 complex was found to have distinct substrate preferences; efficient hydrolysis of Ub dimers with K11-, K48-, and K63-linkages was seen, with weaker hydrolysis observed with K6-, K27-, and K33-linkages and no hydrolysis seen with a K29-linkage. Furthermore, other Ub-like substrates were disfavored by the complex. No activity was seen with SUMO1-GST, SUMO2- and SUMO3-dimers, ISG15-Rho, FAT10-Rho, or Ufm1-Rho, and only weak activity was observed with NEDD8-Rho. Overall, the data presented here characterize the activity and substrate preferences of chUSP1, and thus may facilitate future studies on its in vivo role.

Analysis of the Intracellular Localization of Transiently Expressed and Fluorescently Labeled Copper-Containing Amine Oxidases, Diamine Oxidase and N-Methylputrescine Oxidase in Tobacco, Using an Agrobacterium Infiltration Protocol.

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.

Sequence-structure relationships, expression profiles, and disease-associated mutations in the paralogs of phosphoglucomutase 1.

The key metabolic enzyme phosphoglucomutase 1 (PGM1) controls glucose homeostasis in most human cells. Four proteins related to PGM1, known as PGM2, PGM2L1, PGM3 and PGM5, and referred to herein as paralogs, are encoded in the human genome. Although all members of the same enzyme superfamily, these proteins have distinct substrate preferences and different functional roles. The recent association of PGM1 and PGM3 with inherited enzyme deficiencies prompts us to revisit sequence-structure and other relationships among the PGM1 paralogs, which are understudied despite their importance in human biology. Using currently available sequence, structure, and expression data, we investigated evolutionary relationships, tissue-specific expression profiles, and the amino acid preferences of key active site motifs. Phylogenetic analyses indicate both ancient and more recent divergence between the different enzyme sub-groups comprising the human paralogs. Tissue-specific protein and RNA expression profiles show widely varying patterns for each paralog, providing insight into function and disease pathology. Multiple sequence alignments confirm high conservation of key active site regions, but also reveal differences related to substrate specificity. In addition, we find that sequence variants of PGM2, PGM2L1, and PGM5 verified in the human population affect residues associated with disease-related mutants in PGM1 or PGM3. This suggests that inherited diseases related to dysfunction of these paralogs will likely occur in humans.

A genomic perspective on stoichiometric regulation of soil carbon cycling

Similar to plant growth, soil carbon (C) cycling is constrained by the availability of nitrogen (N) and phosphorus (P). We hypothesized that stoichiometric control over soil microbial C cycling may be shaped by functional guilds with distinct nutrient substrate preferences. Across a series of rice fields spanning 5–25% soil C (N:P from 1:12 to 1:70), C turnover was best correlated with P availability and increased with experimental N addition only in lower C (mineral) soils with N:P⩽16. Microbial community membership also varied with soil stoichiometry but not with N addition. Shotgun metagenome data revealed changes in community functions with increasing C turnover, including a shift from aromatic C to carbohydrate utilization accompanied by lower N uptake and P scavenging. Similar patterns of C, N and P acquisition, along with higher ribosomal RNA operon copy numbers, distinguished that microbial taxa positively correlated with C turnover. Considering such tradeoffs in genomic resource allocation patterns among taxa strengthened correlations between microbial community composition and C cycling, suggesting simplified guilds amenable to ecosystem modeling. Our results suggest that patterns of soil C turnover may reflect community-dependent metabolic shifts driven by resource allocation strategies, analogous to growth rate–stoichiometry coupling in animal and plant communities.

Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.

Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes

Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

Comparison of sampling methodologies and estimation of population parameters for a temporary fish ectoparasite.

Characterizing spatio-temporal variation in the density of organisms in a community is a crucial part of ecological study. However, doing so for small, motile, cryptic species presents multiple challenges, especially where multiple life history stages are involved. Gnathiid isopods are ecologically important marine ectoparasites, micropredators that live in substrate for most of their lives, emerging only once during each juvenile stage to feed on fish blood. Many gnathiid species are nocturnal and most have distinct substrate preferences. Studies of gnathiid use of habitat, exploitation of hosts, and population dynamics have used various trap designs to estimate rates of gnathiid emergence, study sensory ecology, and identify host susceptibility. In the studies reported here, we compare and contrast the performance of emergence, fish-baited and light trap designs, outline the key features of these traps, and determine some life cycle parameters derived from trap counts for the Eastern Caribbean coral-reef gnathiid, Gnathia marleyi. We also used counts from large emergence traps and light traps to estimate additional life cycle parameters, emergence rates, and total gnathiid density on substrate, and to calibrate the light trap design to provide estimates of rate of emergence and total gnathiid density in habitat not amenable to emergence trap deployment.

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design.

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR.