Substrate Cleavage Research Articles

Biophysical characterization and in silico analysis of natural and synthetic compounds targeting Listeria monocytogenes HtrA protease.

HtrA protein is a member of a serine protease family with dual functions as a protease and molecular chaperone. It is a virulence factor in many bacteria, including the food-borne pathogen Listeria monocytogenes (Lm), which induces listeriosis in humans. Hence, inhibitors of LmHtrA protease have great importance in the control of infection. Many natural compounds have been used in the inhibition studies of proteases; here, we have performed the inhibition studies of LmHtrA with 31 compounds from different origins. The spectrophotometric assays revealed that plant compounds are promising inhibitors of LmHtrA protease activity compared to other tested peptides and synthetic compounds. The green tea catechin, EGCG has been identified as an inhibitor of protease activity of LmHtrA with a low IC50 value of 0.754 ± 0.2μM. The substrate cleavage analysis by SDS-PAGE and SPR experiments corroborates the spectrophotometric results by exhibiting protease inhibition and showing the micromolar affinity of EGCG with LmHtrA, respectively. The interaction between rLmHtrA and EGCG was investigated by fluorescence spectroscopy. The binding constant and the number of binding sites were determined as 1.86 × 10(5)M(-1) and 1.2, respectively. The molecular docking and dynamics results of LmHtrA-inhibitor complexes have provided new insights into the inhibition mechanism of LmHtrA compared with other serine proteases. The findings of this study may open up new avenues for the development of natural compound-based derivatives of LmHtrA inhibitors that might be more potent and less harmful to humans.

Identification of pancreatic cancer-specific protease substrates for protease-dependent targeted delivery

Pancreatic ductal adenocarcinoma (PDAC) presents significant challenges due to the inadequacy of existing chemotherapeutics, which often result in toxicity-dependent dose limitations and premature cessation of therapy. Targeted delivery of therapeutic molecules offers a promising solution. Given that PDAC is marked by a desmoplastic reaction with extensive aberrant protease activity, protease-dependent targeted delivery could minimize off-target toxicities and is of increasing interest. The efficacy of targeted delivery hinges on the specificity of the substrates used; insufficient specificity can lead to off-target effects, reducing the advantage over non-targeted methods. Here, we employ an unbiased library approach to screen over 7 million peptide substrates for proteolytic cleavage by PDAC cell lysates, identifying 37 substrates enriched by at least 500-fold after three rounds of selection. As systemically administered targeted delivery depends on the absence of substrate cleavage in circulation, the peptide library was also screened against whole blood lysates, and enriched substrates were removed from the PDAC-enriched dataset to obtain PDAC-specific substrates. In vitro validation using FRET-peptides showed that 13 of the selected 15 substrates are cleaved by a panel of PDAC cell line lysates. Moreover, evaluation against healthy murine organ and human blood lysates to assess off-target cleavage revealed that the identified substrates are indeed PDAC-specific and that several substrates may be superior with respect to PDAC specificity over the CAPN2-responsive substrate, which has recently shown preclinical potential in targeted therapy, but future animal models should address the potential superiority. Overall, we thus identified substrates with high selectivity and sensitivity for PDAC that could be employed in protease-dependent targeted therapies.

Self‐Adaptive Activation of DNAzyme Nanoassembly for Synergistically Combined Gene Therapy

AbstractDNAzyme represents a promising gene silencing toolbox yet is obstructed by the poor substrate accessibility in specific cells. Herein, a compact DNA nanoassembly, incorporating multimeric therapeutic DNAzyme, was prepared for selective delivery of gene‐silencing DNAzyme with requisite cofactors and auxiliary chemo‐drugs. By virtue of the sequence‐conservative duplex‐specific nuclease, the endogenous miRNA catalyzes the successive and site‐specific cleavage of DNA nanoassembly substrate (nominated as the localized RNA walking machine) and thus ensures the liberation/activation of therapeutic agents with high accuracy and efficacy. The miR‐10b‐stimulated DNAzyme was designed to downregulate the TWIST transcription factor, an upstream promotor of miR‐10b, thus acquiring the self‐sufficient downregulation of TWIST/miR‐10b signaling nodes (self‐adaptive negative feedback loop) for abrogating tumor metastasis and chemo‐resistance issues.

Self-Adaptive Activation of DNAzyme Nanoassembly for Synergistically Combined Gene Therapy.

DNAzyme represents a promising gene silencing toolbox yet is obstructed by the poor substrate accessibility in specific cells. Herein, a compact DNA nanoassembly, incorporating multimeric therapeutic DNAzyme, was prepared for selective delivery of gene-silencing DNAzyme with requisite cofactors and auxiliary chemo-drugs. By virtue of the sequence-conservative duplex-specific nuclease, the endogenous miRNA catalyzes the successive and site-specific cleavage of DNA nanoassembly substrate (nominated as the localized RNA walking machine) and thus ensures the liberation/activation of therapeutic agents with high accuracy and efficacy. The miR-10b-stimulated DNAzyme was designed to downregulate the TWIST transcription factor, an upstream promotor of miR-10b, thus acquiring the self-sufficient downregulation of TWIST/miR-10b signaling nodes (self-adaptive negative feedback loop) for abrogating tumor metastasis and chemo-resistance issues.

Spherical DNA Nanomotors Enable Ultrasensitive Detection of Active Enzymes in Extracellular Vesicles for Cancer Diagnosis.

Enzymes encapsulated in extracellular vesicles (EV)s hold promise as biomarkers for early cancer diagnosis. However, precise measurement of their catalytic activities within EVs remains a notable challenge. Here, we report an enzymatically triggered spherical DNA nanomotor (EDM) that enables one-pot, cascaded, and highly sensitive analysis of the activity of EV-associated or free apurinic/apyrimidinic endonuclease 1 (APE1, a key enzyme in base excision repair) across various biological samples. The EDM capitalizes on APE1-triggered activation of DNAzyme (Dz) and its autonomous cleavage of substrates to achieve nonlinear signal amplification. Using EDM, we reveal a strong correlation between APE1 activity in EVs and that of their parental cancer cells. Additionally, EV APE1 mirrors the fluctuation of cellular APE1 activity in response to chemotherapy-induced DNA damage. In a pilot clinical study (n = 63), the EDM-based assay reveals that more than 80% of active APE1 in serum samples is EV-encapsulated. Notably, EV APE1 can differentiate early prostate cancer (PCa) patients from healthy donors (HDs) with an overall accuracy of 92%, outperforming free APE1 in sera. We anticipate that EDM will become a versatile tool for quantifying EV-associated enzymes.

Spherical DNA Nanomotors Enable Ultrasensitive Detection of Active Enzymes in Extracellular Vesicles for Cancer Diagnosis

Enzymes encapsulated in extracellular vesicles (EV)s hold promise as biomarkers for early cancer diagnosis. However, precise measurement of their catalytic activities within EVs remains a notable challenge. Here, we report an enzymatically triggered spherical DNA nanomotor (EDM) that enables one‐pot, cascaded, and highly sensitive analysis of the activity of EV‐associated or free apurinic/apyrimidinic endonuclease 1 (APE1, a key enzyme in base excision repair) across various biological samples. The EDM capitalizes on APE1‐triggered activation of DNAzyme (Dz) and its autonomous cleavage of substrates to achieve nonlinear signal amplification. Using EDM, we reveal a strong correlation between APE1 activity in EVs and that of their parental cancer cells. Additionally, EV APE1 mirrors the fluctuation of cellular APE1 activity in response to chemotherapy‐induced DNA damage. In a pilot clinical study (n = 63), the EDM‐based assay reveals that more than 80% of active APE1 in serum samples is EV‐encapsulated. Notably, EV APE1 can differentiate early prostate cancer (PCa) patients from healthy donors (HDs) with an overall accuracy of 92%, outperforming free APE1 in sera. We anticipate that EDM will become a versatile tool for quantifying EV‐associated enzymes.

Biochemical characterization and structure prediction of the Cerrado soil CRB2(1) metagenomic dioxygenase

Dioxygenases are enzymes involved in the conversion of polyconic aromatic hydroxycarbons (PAHs), attracting significant biotechnological interest for the conversion of recalcitrant organic compounds. Furthermore, few studies show that dioxygenases can take on the function of resistance genes in clones. This enzymatic versatility opens up new opportunities for elucidating the mechanisms of microbial resistance, as well as its biotechnological application. In this work, a Cerrado soil dioxygenase named CRB2(1) was biochemically characterized. The enzyme was shown to have optimal activity at pH 7; a temperature of 30 °C; and using iron ions as a cofactor for substrate cleavage. The kinetic catalytic parameters of CRB2(1) were Vmax = 0.02281 µM/min and KM = 97.6. Its predicted three-dimensional structure obtained using the Modeller software v9.22 based on the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) (PDB ID 3BU7, resolution 2.80 Å, residues 17–374) revealed substrate binding to the cupin domain, where the active site is located. The analyzed substrates interact directly with the iron ion, coordinated by three histidine residues. Changing the iron ion charge modifies the binding between the active site and the substrates. Currently, there is a demand for enzymes that have biotechnological activities of interest. Metagenomics allows analyzing the biotechnological potential of several organisms at the same time, based on sequence and functional activity analyses.

Hydrogen Sulfide‐Triggered Artificial DNAzyme Switches for Precise Manipulation of Cellular Functions

AbstractThe development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H2S). To address this, we present the design of Gas signaling molecule‐Responsive Artificial DNAzyme‐based Switches (GRAS) to manipulate cellular functions via H2S‐sensitive synthetic DNAzymes. By incorporating stimuli‐responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H2S‐responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H2S‐reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H2S‐induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H2S‐triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering.

SERRATE drives phase separation behaviours to regulate m6A modification and miRNA biogenesis.

The methyltransferase complex (MTC) deposits N6-adenosine (m6A) onto RNA, whereas the microprocessor produces microRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B tends to form insoluble condensates with poor activity, with its level monitored by the 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promote the solubility and stability of the MTC subunit B, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behaviour, exhibit reduced m6A levels. Reciprocally, MTC can recruit the microprocessor to the MIRNA loci, prompting co-transcriptional cleavage of primary miRNA substrates. Additionally, primary miRNA substrates carrying m6A modifications at their single-stranded basal regions are enriched by m6A readers, which retain the microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis.

A strategy for detecting CSFV using DNAzyme-HCR cascade amplification.

The Hybridization Chain Reaction (HCR) is an isothermal amplification technique widely used for sensing nucleic acids and small molecules. Despite its effectiveness, conventional linear HCR exhibits relatively slow kinetics and insufficient sensitivity. To address this challenge, we have innovatively combined HCR with DNAzyme technology to enhance nucleic acid detection. In this novel approach, the presence of a target molecule triggers the formation of DNAzyme, leading to the cleavage of substrate S, the initiation of HCR, and the production of DNA nanowires and labeled DNAzyme. The newly generated DNAzyme continuously cleaves substrate S, promoting sequential HCR amplification and significantly enhancing the fluorescence signal. This system offers a simple, sensitive, selective, and versatile method for nucleic acid detection, with a detection limit as low as 5 pM. When tested on classical swine fever virus (CSFV) samples, the system demonstrated detection accuracy comparable to RT-qPCR and exhibited superior repeatability.

Cleavage of DNA Substrate Containing Nucleotide Mismatch in the Complementary Region to sgRNA by Cas9 Endonuclease: Thermodynamic and Structural Features

The non-ideal accuracy and insufficient selectivity of CRISPR/Cas9 systems is a serious problem for their use as a genome editing tool. It is important to select the target sequence correctly so that the CRISPR/Cas9 system does not cut similar sequences. This requires an understanding of how and why mismatches in the target sequence can affect the efficiency of the Cas9/sgRNA complex. In this work, we studied the catalytic activity of the Cas9 enzyme to cleave DNA substrates containing nucleotide mismatch at different positions relative to the PAM in the “seed” sequence. We show that mismatches in the complementarity of the sgRNA/DNA duplex at different positions relative to the protospacer adjacent motif (PAM) sequence tend to decrease the cleavage efficiency and increase the half-maximal reaction time. However, for two mismatches at positions 11 and 20 relative to the PAM, an increase in cleavage efficiency was observed, both with and without an increase in half-reaction time. Thermodynamic parameters were obtained from molecular dynamics results, which showed that mismatches at positions 8, 11, and 20 relative to the PAM thermodynamically stabilize the formed complex, and a mismatch at position 2 of the PAM fragment exerts the greatest stabilization compared to the original DNA sequence. The weak correlation of the thermodynamic binding parameters of the components of the Cas9/sgRNA:dsDNA complex with the cleavage data of DNA substrates containing mismatches indicates that the efficiency of Cas9 operation is mainly affected by the conformational changes in Cas9 and the mutual arrangement of sgRNA and substrates.

Stabilization of the open conformation οf insulin-regulated aminopeptidase by a novel substrate-selective small-molecule inhibitor.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

Label-free SELEX of aptamers for ultra-sensitive electrochemical aptasensor detection of amanitin in wild mushrooms

BackgroundMushroom poisoning poses a significant global health concern, with high morbidity and mortality rates. The primary lethal toxins responsible for this condition are alpha-amanitin (ɑ-AMA) and beta-amanitin (β-AMA). As a promising bio-recognition molecules in biosensors, aptamers, have been broadly used in the field of food detection. However, the current SELEX-based methods for screening aptamers for structurally similar small molecules were limited by the labelling or salt ion induction. In this study, we aimed to develop a novel label-free SELEX strategy for the screening of aptamers with high affinity and constructed new aptasensors for the detection of ɑ-AMA and β-AMA. ResultsA novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for ɑ-AMA and β-AMA. Only 18 rounds of SELEX were required to obtain new aptamers. The candidate aptamers were analyzed by colloidal gold assay, and the sequences of ɑ-30 and β-37 displayed great affinity with Kd values of 22.26 nM and 23.32 nM, respectively, without interference from botanical toxins. Notably, the truncated aptamers ɑ-30-2 (50 bp) and β-37-2 (57 bp) exhibited higher affinity than their original counterpart (79 bp). Subsequently, the selected aptamers were utilized to construct recognition probes for electrochemical aptasensors based on hairpin cyclic cleavage of substrates by Cu2+ dependent DNAzyme and Exo I-triggered recycling cascades. The detection platform showed excellent analytical performance with limits of detection as low as 4.57 pg/mL (ɑ-AMA) and 8.49 pg/mL (β-AMA). Moreover, the aptasensors exhibited superior performance in mushroom and urine samples. SignificanceThis work developed a simple and efficient label-free SELEX method for screening new aptamers for ɑ-AMA and β-AMA, which employed the positively charged AuNPs as the screening medium, without the need for chemical labelling of libraries or induction of salt ions. Furthermore, two novel electrochemical aptasensors were developed based on our newly obtained aptamers, which offer the new biosensing tool for ultrasensitive detection of the AMA poisoning, showing great potential in practical applications.

Chlamydia-containing spheres are a novel and predominant form of egress by the pathogen Chlamydia psittaci.

The egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci, little is known about the mechanisms resulting in bacterial egress from the infected epithelium. Here, we describe and characterize Chlamydia-containing spheres (CCSs), a novel and predominant type of non-lytic egress utilized by Chlamydia spp. CCSs are spherical, low-phase contrast structures surrounded by a phosphatidylserine-exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. CCS formation is a sequential process starting with the proteolytic cleavage of a DEVD tetrapeptide-containing substrate that can be detected inside the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected cell. Subsequently, blebbing of the plasma membrane begins, the inclusion membrane destabilizes, and the proteolytic cleavage of a DEVD-containing substrate increases rapidly within the whole infected cell. Finally, infected, blebbing cells detach and leave the monolayer, thereby forming CCS. This sequence of events is unique for chlamydial CCS formation and fundamentally different from previously described Chlamydia egress pathways. Thus, CCS formation represents a major, previously uncharacterized egress pathway for intracellular pathogens that could be linked to Chlamydia biology in general and might influence the infection outcome in vivo.IMPORTANCEHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host transmission. Here, we characterize Chlamydia-containing sphere (CCS) formation as a novel and predominant non-lytic egress pathway of the intracellular pathogens Chlamydia psittaci and Chlamydia trachomatis. CCS formation is fundamentally different from extrusion formation, the previously described non-lytic egress pathway of C. trachomatis. CCS formation is a unique sequential process, including proteolytic activity, followed by an increase in intracellular calcium concentration, inclusion membrane destabilization, plasma membrane blebbing, and the final detachment of a whole phosphatidylserine-exposing former host cell. Thus, CCS formation represents an important and previously uncharacterized egress pathway for intracellular pathogens that could possibly be linked to Chlamydia biology, including host tropism, protection from host cell defense mechanisms, or bacterial pathogenicity.

Structural basis for the activity of the type VII CRISPR-Cas system.

The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA1. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5' end, but not at the 3' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications.

Two RNA Folds from One Sequence: A Ribozyme with Versatile Substrate Processing Abilities.

We report the design of a single RNA sequence capable of adopting one of two ribozyme folds and catalyzing the cleavage and/or ligation of the respective substrates. The RNA is able to change its conformation in response to its environment, hence it is called chameleon ribozyme (CHR). Efficient RNA cleavage of two different substrates as well as RNA ligation by CHR is demonstrated in separate experiments and in a one pot reaction. Our study shows that sequence variants of the hairpin ribozyme intersect with the hammerhead ribozyme and that rather short RNA molecules can have comprehensive conformational flexibility, which is an important feature for the emergence of new functional folds in early evolution.

Membrane Localization of RNase Y Is Important for Global Gene Expression in Bacillus subtilis.

RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.

Identification of a novel caspase cleavage motif AEAD

Infections of many viruses induce caspase activation to regulate multiple cellular pathways, including programmed cell death, immune signaling and etc. Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation. Here, we identified and analyzed a novel caspase cleavage motif AEAD, and confirmed its caspase dependent cleavage activity in natural substrate, such as nitric oxide-associated protein 1 (NOA1). Fusing the enhanced green fluorescent protein (EGFP) with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria. Upon the activation of caspase triggered by Sendai virus (SeV) or herpes simplex virus type 1 (HSV-1) infection, EGFP diffusely localized to the cell due to the caspase-mediated cleavage, thus allowing visual detection of the virus-induced caspase activation. An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed, which significantly inhibited the activities of caspases-1, -3, -6, -7, -8 and -9, exhibiting a broad caspase inhibition effect. The inhibitor further prevented caspases-mediated cleavage of downstream substrates, including BID, PARP1, LMNA, pro-IL-1β, pro-IL-18, GSDMD and GSDME, protecting cells from virus-induced apoptotic and pyroptotic cell death. Together, our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.

Abstract Tu129: Collagen Peptide modulates Enzymatic Activity post-Myocardial Infarction

Matrix metalloproteinases (MMPs) play critical roles post-myocardial infarction (MI) and directly correlate with adverse LV remodeling. Of the MMPs, MMP-9 shows the highest correlation with MI patient mortality. Clinical trials trying to inhibit MMP-9 were unsuccessful or detrimental, mostly because MMP-9 has critical roles post-MI and inhibition results in exacerbation of adverse remodeling. Previously, we reported a collagen peptide – p1159 – to reduce LV dilation and adverse LV remodeling post-MI. Recently, we discovered p1159 acts as an MMP-9 competitive substrate and reduces substrate proteolysis in vitro . Herein, we investigated whether p1159 can act as a competitive substrate in vivo to reduce proteolysis of endogenous substrates post-MI without inhibiting MMP-9 activity. Also, we compared p1159 actions to those of a MMP-9 inhibitor (MMP-9i). We hypothesized that animals that receive p1159 would show reduced inflammation and adverse remodeling compared to those that received MMP-9i. Adult male and female C57Bl/6 mice (n = 6-8/group) were grouped as follows: saline (control); p1159; MMP-9i, and p1159 + MMP-9i. We performed baseline echocardiography followed by surgery to induce a permanent occlusion MI model. Three hours later, mice received a randomized osmotic mini-pump to continuously deliver p1159 (14μg/day/kg body weight) or vehicle (saline, 0.9%) and/or MMP-9i (10mg/kg body weight, JNJ 0966 by gavage). The LV and plasma were harvested on day 3 (D3) and D5 for downstream proteomics analysis following a final echocardiographic assessment. Infarct sizes were similar between groups, indicating same level of initial injury. Compared to control, p1159 did not inhibit activity or levels of MMP-9, while animals treated with MMP-9i showed significantly reduced MMP-9 activity. As expected, p1159 reduced (p < 0.05) substrate cleavage, as seen by analysis of the LV peptidome, particularly at D3. Substrates that displayed a reduction in proteolysis included fibrinogen, MIF, myosin, titin, and tubulin. These data support p1159 as a competitive substrate of MMP-9 that can modulate its activity without inhibition. Moreover, these results may explain the previously reported effect of p1159 at reducing LV dilation and improving systolic function.