Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Tailored Branched Polymer-Protein Bioconjugates for Tunable Sieving Performance.

Protein-polymer conjugates combine the unique properties of both proteins and synthetic polymers, making them important materials for biomedical applications. In this work, we synthesized and characterized protein-branched polymer bioconjugates that were precisely designed to retain protein functionality while preventing unwanted interactions. Using chymotrypsin as a model protein, we employed a controlled radical branching polymerization (CRBP) technique utilizing a water-soluble inibramer, sodium 2-bromoacrylate. The green-light-induced atom transfer radical polymerization (ATRP) enabled the grafting of branched polymers directly from the protein surface in the open air. The resulting bioconjugates exhibited a predetermined molecular weight, well-defined architecture, and high branching density. Conformational analysis by SEC-MALS validated the controlled grafting of branched polymers. Furthermore, enzymatic assays revealed that densely grafted polymers prevented protein inhibitor penetration, and the resulting conjugates retained up to 90% of their enzymatic activity. This study demonstrates a promising strategy for designing protein-polymer bioconjugates with tunable sieving behavior, opening avenues for applications in drug delivery and biotechnology.

Therapeutic potential of proteases in acute lung injury and respiratory distress syndrome via TLR4/Nrf2/NF-kB signaling modulation.

The COVID-19 pandemic has drawn attention to acute lung injury and respiratory distress syndrome as major causes of death, underscoring the urgent need for effective treatments. Protease enzymes possess a wide range of beneficial effects, including antioxidant, anti-inflammatory, antifibrotic, and fibrinolytic effects. This study aimed to evaluate the potential therapeutic effects of bacterial protease and chymotrypsin in rats in mitigating acute lung injury induced by lipopolysaccharide. Molecular docking was employed to investigate the inhibitory effect of bacterial protease and chymotrypsin on TLR-4, the receptor for lipopolysaccharide. Bacterial protease restored TLR-4, Nrf2, p38 MAPK, NF-kB, and IKK-ß levels to normal levels, while chymotrypsin normalized TLR-4, IKK-ß, IL-6, and IL-17 levels. The expression of TGF-ß, caspase-3, and VEGF in the bacterial protease- and chymotrypsin-treated groups was markedly reduced. Our results suggest that both therapies ameliorate LPS-induced acute lung injury and modulate the TLR4/Nrf2/NF-k signaling pathway. Each protease exhibited distinct mechanisms, with bacterial protease showing a better response to oxidative stress, edema, and fibrosis, whereas chymotrypsin provided a better response in the acute phase and innate immunity. These findings highlight the potential of each protease as a promising therapeutic option for acute lung injury and respiratory distress syndrome.

[Effects of removing superficial layer of cartilage on the surface morphology and mechanical behavior of cartilage].

Superficial cartilage defect is an important factor that causes osteoarthritis. Therefore, it is very important to investigate the influence of superficial cartilage defects on its surface morphology and mechanical properties. In this study, the knee joint cartilage samples of adult pig were prepared, which were treated by enzymolysis with chymotrypsin and physical removal with electric friction pen, respectively. Normal cartilage and surface treated cartilage were divided into five groups: control group (normal cartilage group), chymotrypsin immersion group, chymotrypsin wiping group, removal 10% group with electric friction pen, and removal 20% group with electric friction pen. The surface morphology and structure of five groups of samples were characterized by laser spectrum confocal microscopy and environmental field scanning electron microscopy, and the mechanical properties of each group of samples were evaluated by tensile tests. The results show that the surface arithmetic mean height and fracture strength of the control group were the smallest, and the fracture strain was the largest. The surface arithmetic mean height and fracture strength of the removal 20% group with electric friction pen were the largest, and the fracture strain was the smallest. The surface arithmetic mean height, fracture strength and fracture strain values of the other three groups were all between the above two groups, but the surface arithmetic mean height and fracture strength of the removal 10% group with electric friction pen, the chymotrypsin wiping group and the chymotrypsin soaking group decreased successively, and the fracture strain increased successively. In addition, we carried out a study on the elastic modulus of different groups, and the results showed that the elastic modulus of the control group was the smallest, and the elastic modulus of the removal 20% group with electric friction pen was the largest. The above study revealed that the defect of the superficial area of cartilage changed its surface morphology and structure, and reduced its mechanical properties. The research results are of great significance for the prevention and repair of cartilage injury.

A Multicomponent Mannich Reaction Catalyzed by Hydrolases Immobilized on Titanate Nanotubes.

This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

Thrombin has dual trypsin-like and chymotrypsin-like specificity.

BACKGROUND: The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVES: To establish if thrombin can cleave at Trp residues. METHODS: The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage. RESULTS: Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSION: The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

Risk of chronic pancreatitis in carriers of the c.180C>T (p.Gly60=) CTRC variant: case-control studies and meta-analysis.

Chymotrypsin C (CTRC) is a digestive serine protease produced by the pancreas that regulates intrapancreatic trypsin activity and provides a defensive mechanism against chronic pancreatitis (CP). CTRC exerts its protective effect by promoting degradation of trypsinogen, the precursor to trypsin. Loss-of-function missense and microdeletion variants of CTRC are found in around 4% of CP cases and increase disease risk by approximately 3-7-fold. In addition, a commonly occurring synonymous CTRC variant c.180C>T (p.Gly60=) was reported to increase CP risk in various cohorts but a global analysis of its impact has been lacking. Here, we analyzed the frequency and effect size of variant c.180C>T in Hungarian and pan-European cohorts, and performed meta-analysis of the new and published genetic association data. When allele frequency was considered, meta-analysis revealed an overall frequency of 14.2% in patients and 8.7% in controls (allelic odds ratio (OR) 2.18, 95% confidence interval (CI) 1.72-2.75). When genotypes were examined, c.180TT homozygosity was observed in 3.9% of CP patients and in 1.2% of controls, and c.180CT heterozygosity was present in 22.9% of CP patients and in 15.5% of controls. Relative to the c.180CC genotype, the genotypic OR values were 5.29 (95% CI 2.63-10.64), and 1.94 (95% CI 1.57-2.38), respectively, indicating stronger CP risk in homozygous carriers. Finally, we obtained preliminary evidence that the variant is associated with reduced CTRC mRNA levels in the pancreas. Taken together, the results indicate that CTRC variant c.180C>T is a clinically relevant risk factor, and should be considered when genetic etiology of CP is investigated.

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2.

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15 N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics, and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1°C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes.

A meta-analysis for assessing the contributions of trypsin and chymotrypsin as the two major endoproteases in protein hydrolysis in fish intestine.

For the majority of fish species, regardless of being gastric or agastric, trypsin and chymotrypsin are known as the two main alkaline proteases responsible for the initial stage of protein hydrolysis in the fish intestine. Although the critical role of these proteases for protein hydrolysis in fish intestine is without doubt, the relative input of each enzyme in protein hydrolysis is still unclear. Data used in the present study has been retrieved from a bibliographic search using the Dimensions application (https://app.dimensions.ai/discover/publication tool). Retrieved articles were carefully inspected to identify whether they contained the description of the development of ontogenetic activities for trypsin, chymotrypsin, and total alkaline proteases in fish intestine. From the list of consulted articles, 21 studies were chosen based on correlation coefficients (Pearson correlation test), and four groups of fish were identified with high significant correlation between 1) the activity of chymotrypsin and total alkaline proteases; 2) the activity of trypsin, chymotrypsin, and total alkaline proteases; 3) the activity of trypsin and total alkaline proteases, and 4) mainly negative correlation between trypsin, chymotrypsin, and total alkaline proteases. These results indicated that the relative inputs of trypsin and chymotrypsin in protein hydrolysis may vary significantly among different fish species, which is a crucial point for proper understanding of species-specific digestive traits in both natural and aquaculture scenarios.

Variants in KLK11, affecting signal peptide cleavage of kallikrein-related peptidase 11, cause an autosomal-dominant cornification disorder.

BACKGROUND: Mendelian disorders of cornification (MeDOC) are a group of heterogeneous genodermatoses with different genetic bases. The pathogenesis of a substantial group of MeDOC remains to be elucidated. OBJECTIVES: To identify a new causative gene and the pathogenesis of a previously undescribed autosomal-dominant cornification disorder. METHODS: Whole-exome sequencing was performed in three families with the novel cornification disorder to identify the disease-causing variants. As the variants were located around the signal peptide (SP) cleavage site of a kallikrein-related peptidase, SP cleavage, subcellular localization and extracellular secretion of the variants were evaluated in eukaryotic overexpression systems by Western blotting or immunocytochemistry. Then the trypsin-like and chymotrypsin-like proteolytic activity of the peptidase and degradation of its catalytic substrate were assayed using the patients' stratum corneum (SC) samples. The morphology of the lamellar bodies and corneodesmosomes (CDs) in the patients' SC was ultrastructurally examined. A mouse model harbouring the equivalent variant was constructed and evaluated histologically. RESULTS: We identified two heterozygous variants affecting Gly50 in kallikrein-related peptidase (KLK)11 in a familial case and two sporadic cases with the new disorder, which is characterized by early-onset ichthyosiform erythroderma or erythrokeratoderma. KLK11 belongs to the family of kallikrein-related peptidases participating in skin desquamation by decomposing CDs, a process essential for shedding of the SC. In vitro experiments demonstrated that the variants perturbed the SP cleavage of KLK11, leading to subcellular mislocalization and impaired extracellular secretion of the KLK11 Gly50Glu variant. Both trypsin-like and chymotrypsin-like proteolytic activities were significantly decreased in the patients' SC samples. Reduced proteolysis of desmoglein 1 and delayed degeneration of CDs were detected in patients' SC, indicating delayed skin desquamation. Consistently, the patients showed a thickened, dense SC, indicating abnormal skin desquamation. Mice harbouring the homozygous c.131G>A (p.Gly44Glu) Klk11 variant, which is equivalent to KLK11 c.149G>A (p.Gly50Glu) in humans, exhibited hyperkeratosis and abnormal desquamation, partially recapitulating the phenotype. CONCLUSIONS: We provide evidence that variants at Gly50 affecting the SP cleavage of KLK11 cause a new autosomal-dominant cornification disorder with abnormal desquamation. Our findings highlight the essential role of KLKs in maintaining homeostasis of skin keratinization and desquamation.

Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

Proteomic Analysis Identifies Three Reliable Biomarkers of Intestinal Inflammation in the Stools of Patients With Inflammatory Bowel Disease.

BACKGROUND: Faecal biomarkers have emerged as important tools in managing of inflammatory bowel disease [IBD], which includes Crohn's disease [CD] and ulcerative colitis [UC]. AIM: To identify new biomarkers of gut inflammation in the stools of IBD patients using a proteomic approach. METHODS: Proteomic analysis of stools was performed in patients with both active CD and CD in remission and in controls by 2-DIGE and MALDI-TOF/TOF MS. An ELISA was used to confirm results in a second cohort of IBD patients and controls. RESULTS: 2-DIGE analysis detected 70 spots in the stools of patients with active CD or patients in remission CD and in controls. MALDI-TOF/TOF MS analysis identified 21 proteins with Chymotrypsin C, Gelsolin and Rho GDP-dissociation inhibitor 2 [RhoGDI2] best correlating with the levels of intestinal inflammation. Results were confirmed in a second cohort of IBD patients and controls [57 CD, 60 UC, 31 controls]. The identified faecal markers significantly correlated with the severity of intestinal inflammation in IBD patients [SES-CD in CD, Mayo endoscopic subscore in UC] [CD; Chymotrypsin-C: r = 0.64, p < 0.001; Gelsolin: r = 0.82, p < 0.001; RhoGDI2: r = 0.64, p < 0.001; UC; Chymotrypsin-C: r = 0.76, p < 0.001; Gelsolin: r = 0.75, p < 0.001; RhoGDI2: r = 0.63, p < 0.001]. Moreover, ROC analysis showed that Gelsolin [p < 0.0002] and RhoGDI2 [p < 0.0001] in CD, and RhoGDI2 [p = 0.0004] in UC, have higher sensitivity and specificity than faecal calprotectin in discriminating between patients and controls. CONCLUSIONS: We show for the first time that 2-DIGE is a reliable method to detect proteins in human stools. Three novel faecal biomarkers of gut inflammation have been identified that display good specificity and sensitivity for identifying IBD and significantly correlate with IBD severity.

De Novo Design of a Highly Selective Nonpeptide Fluorogenic Probe for Chymotrypsin Activity Sensing in a Living System.

Chymotrypsin, an extensively known proteolytic enzyme, plays a substantial role in maintaining physiological functions, including protein digestion, immune response, and tissue repair. To date, intense attention has been focused on the invention of efficient and sensitive chemical tools for chymotrypsin activity measurement. Among them, the "nonpeptide"-based chymotrypsin probe design strategy utilizing the esterase activity of chymotrypsin has been well-developed due to its low cost and high atom-economy feature. However, the ester-bond-based nature of these probes make them possibly vulnerable to esterases and active chemicals. These defects strictly restricted the application of the previously reported probes, especially for imaging in living systems. Therefore, to acquire fluorogenic probes with sufficient stability and specificity for chymotrypsin sensing in a complicated biological environment, a more stable skeleton for nonpeptide-based chymotrypsin probe construction is urgently needed. Herein, a novel nonpeptide-based fluorogenic probe for specific chymotrypsin activity sensing was designed and synthesized by the substitution of an ester-based linker with a heptafluorobutylamide moiety. The acquired probe, named TMBIHF, showed high selectivity toward various enzymes and reactive chemicals, while it retained high sensitivity and catalytic efficiency toward chymotrypsin. Moreover, TMBIHF was successfully applied for monitoring chymotrypsin activity and pancreas development in live zebrafish, specific sensing of exogenous and endogenous chymotrypsin in nude mice, and visualizing chymotrypsin-like activity-dependent cellular apoptosis, thus providing an alternative and reliable way for chymotrypsin-targeted biosensor or prodrug construction.

Adaptation of Helicoverpa armigera to Soybean Peptidase Inhibitors Is Associated with the Transgenerational Upregulation of Serine Peptidases.

Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis.

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

Development of novel enzyme immobilization methods employing formaldehyde or triethoxysilylbutyraldehyde to fabricate immobilized enzyme microreactors for peptide mapping.

The digestion of proteins with proteolytic enzymes has expedited the analysis of peptide mapping. Here, we compared the digestion efficiency of soluble chymotrypsin (CT) with two immobilized CT preparations using bovine serum albumin (BSA) as the substrate. An efficient method of immobilizing chymotrypsin using formaldehyde (FA) was optimized and the conditions were applied to assess a novel immobilization reagent, triethoxysilylbutaraldehyde (TESB). Efforts to determine the best enzyme-to-substrate (E : S) ratios during digestion of denatured BSA with single-use FA-CT enzyme particles were performed by adjusting the amount of substrate used. An E : S ratio of 10 : 1 was found to be best based on the LC-MS/MS analysis data showing sequence coverage of 67%. Fabrication of immobilized enzyme microreactors (IMERs) was carried out using both (3-aminopropyl)triethoxysilane (APTES) with the idealized conditions with FA, as well as the novel procedure utilizing TESB for a proof of concept open-tubular IMER. It was found that the FA-APTES IMER had a sequence coverage of 6%, while the TESB IMER had 29% sequence coverage from MS analysis. The application of TESB in enzyme immobilization has the potential to facilitate a greater degree of enzymatic digestion with higher sequence coverage than traditional immobilization or crosslinking reagents for bottom-up proteomics.

Identification and validation of two alternatively spliced novel isoforms of human α-1-antichymotrypsin.

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5' and 3' region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of ß-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.

Protein Myristoylation Plays a Role in the Nuclear Entry of the Parvovirus Minute Virus of Mice.

Being nonpathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses with great potential as anti-cancer agents. As these viruses replicate in the host cell nucleus, they must gain access to the nucleus during infection. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the nuclear envelope (NE) and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. In this study, we used MVM as a model to investigate the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays, mutagenesis, and pharmacological inhibition, we identified a functional myristoylation site at the sequence 78GGKVGH83 of the unique portion of the capsid protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this myristoylation site or with purified virions, we found tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying this cleavage exposes G (glycine) 78 at the N-terminus of VP1u for myristoylation. Subsequent experiments using inhibitors of myristoylation and cellular proteases with MVM-infected cells, or an imaging-based quantitative NE permeabilization assay, further indicate protein myristoylation and a chymotrypsin-like activity are essential for MVM to locally disrupt the NE during viral nuclear entry. We thus propose a model for the nuclear entry of MVM in which NE disruption is mediated by VP1u myristoylation after the intact capsid undergoes proteolytic processing to expose the required N-terminal G for myristoylation. IMPORTANCE Rodent parvoviruses (PVs), including minute virus of mice (MVM), have the ability to infect and kill cancer cells and thereby possess great potential in anti-cancer therapy. In fact, some of these viruses are currently being investigated in both preclinical studies and clinical trials to treat a wide variety of cancers. However, the detailed mechanism of how PVs enter the cell nucleus remains unknown. In this study, we for the first time demonstrated a chemical modification called "myristoylation" of a MVM protein plays an essential role in the nuclear entry of the virus. We also showed, in addition to protein myristoylation, a chymotrypsin-like activity, which may come from cellular proteasomes, is required for MVM to get myristoylated and enter the nucleus. These findings deepen our understanding on how MVM and other related PVs infect host cells and provide new insights for the development of PV-based anti-cancer therapies.