Cleavage Events Research Articles

Functional Analysis of Promoters, mRNA Cleavage, and mRNA Secondary Structure on esxB-esxA in Mycolicibacterium smegmatis

The ESX-1 secretion system is critical for the virulence of Mycobacterium tuberculosis as well as for conjugation in the saprophytic model Mycolicibacterium smegmatis. EsxB (CFP-10) and EsxA (ESAT-6) are secreted effectors required for the function of ESX-1 systems. While some transcription factors regulating the expression of esxB and esxA have been identified, little work has addressed their promoter structures or other determinants of their expression. Here, we defined two promoters, one located two genes upstream of esxB and one located immediately upstream, that contribute substantially to the expression of esxB and esxA. We also defined an mRNA cleavage site within the esxB 5′ untranslated region (UTR) and found that a single-nucleotide substitution reprogramed the position of this cleavage event without impacting esxB-esxA transcript abundance. We furthermore investigated the impact of a double stem-loop structure in the esxB 5′ UTR and found that it does not confer stability on a reporter gene transcript. Consistent with this, there was no detectable correlation between mRNA half-life and secondary structure near the 5′ ends of 5′ UTRs on a transcriptome-wide basis. Collectively, these data shed light on the determinants of esxB-esxA expression in M. smegmatis as well as provide broader insight into the determinants of mRNA cleavage in mycobacteria and the relationship between 5′ UTR secondary structure and mRNA stability.

Functional and molecular insights into the role of Sae2 C-terminus in the activation of MRX endonuclease.

The yeast Sae2 protein, known as CtIP in mammals, once phosphorylated at Ser267, stimulates the endonuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex to cleave DNA ends that possess hairpin structures or protein blocks, such as the Spo11 transesterase or trapped topoisomerases. Stimulation of the Mre11 endonuclease by Sae2 depends on a Rad50-Sae2 interaction, but the mechanism by which this is achieved remains to be elucidated. Through genetic studies, we show that the absence of the last 23 amino acids from the Sae2 C-terminus specifically impairs MRX-dependent DNA cleavage events, while preserving the other Sae2 functions. Employing AlphaFold3 protein structure predictions, we found that theRad50-Sae2 interface involves not only phosphorylated Ser267 but also the phosphorylated Thr279 residue and the C-terminus of Sae2. This region engages in multiple interactions with residues that are mutated in rad50-s mutants, which are known to be specifically defective in the processing of Spo11-bound DNA ends. These interactions are critical for stabilizing the association between Sae2 and Rad50, thereby ensuring the correct positioning of Mre11 in its active endonucleolytic state.

Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry.

Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

CIRCLE-Seq for Interrogation of Off-Target Gene Editing.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

PlmCas12e Utilizes Glu662 to Prevent Cleavage Site Occupation by Positively Charged Residues Before Target Strand Cleavage.

CRISPR-Cas12e is a recently identified gene-editing tool mainly known because its relatively small size benefits cell delivery. Drastically different from Cas9, it creates a blunt-end double-strand breakage of the DNA via two cleavage sites; Cas12e produces a sticky-end double-strand breakage of the DNA through only one cleavage site in its RuvC domain, meaning two consecutive cleavage events first on the non-target strand (ntsDNA) and then the target strand (tsDNA). Though crucial for Cas12e's cleavage efficiency, the mechanism by which Cas12e loads tsDNA for the second cleavage remains elusive. Through molecular dynamics simulations and our recently matured traveling-salesman-based automated path-searching (TAPS) algorithm, we identified a series of positively charged residues (Arg856TSL, Arg768RuvC, Lys898TSL, Arg904TSL, Arg764RuvC) that guide the tsDNA backbone toward the cleavage site of wild-type PlmCas12e. Further simulations of the R856L and R904L mutants supported such observations. More interestingly, we found the key role of Glu662RuvC in coordinating Arg764RuvC, preventing its occupation of the cleavage site, and facilitating tsDNA cleavage. Additional simulations confirmed that mutating Glu662RuvC to valine disabled such coordination and created a stable intermediate state with Arg764RuvC occupying the cleavage site before tsDNA loading. These insights, revealing an elaborate mechanism of cleavage facilitation, offer essential guiding principles for future rational engineering of Cas12e into more efficient gene-editing tools.

The kinetics of nucleolar precursor bodies clustering at the pronuclei interface: Positive correlations with the morphokinetic characteristics of cleaving embryos and euploidy in preimplantation genetic testing programs

Objective: This study investigated potential relationships between the kinetics of nucleolar precursor bodies (NPBs) in the pronucleus and developmental morphokinetics and euploidy in human preimplantation genetic testing for aneuploidy (PGT-A) cycles.Methods: The morphokinetic analysis of 200 blastocysts obtained from 53 PGT-A cycles was performed retrospectively in a time-lapse incubator. At the time of pronuclear breakdown (PNBD), we categorized the blastocysts into two groups based on the kinetic degree of clustering NPBs at the interface of the two pronuclei: clustered NPBs (CL) and non-clustered NPBs (NCL). We then compared morphokinetic parameters, abnormal behavioral events, and the rate of aneuploidy between the two groups.Results: Pronuclear fading and the first cleavage occurred earlier in the NCL group than in the CL group. However, the initiation of blastocyst formation and blastocyst expansion was delayed in the NCL group relative to the CL group. No differences were found in the rate of abnormal cleavage events, such as multinucleation at the 2-cell stage, direct cleavage from one to three cells, and from two to five cells between the CL and NCL groups. However, the fragmentation rate at the 8-cell stage was higher in the NCL group than in the CL group (10.3% vs. 1.9%, p<0.05). Additionally, the euploid rate in the CL group was significantly higher than in the NCL group (37.9% vs. 12.4%, p<0.05).Conclusion: These results demonstrate the effectiveness of combining NPB clustering at PNBD with morphokinetics as a parameter for selecting embryos with higher developmental potential in in vitro fertilization.

Histone Tail Cleavage as a Mechanism for Epigenetic Regulation.

Histones are essential for DNA packaging and undergo post-translational modifications that significantly influence gene regulation. Among these modifications, histone tail cleavage has recently garnered attention despite being less explored. Cleavage by various proteases impacts processes such as stem cell differentiation, aging, infection, and inflammation, though the mechanisms remain unclear. This review delves into recent insights on histone proteolytic cleavage and its epigenetic significance, highlighting how chromatin, which serves as a dynamic scaffold, responds to signals through histone modification, replacement, and ATP-dependent remodeling. Specifically, histone tail cleavage is linked to critical cellular processes such as granulocyte differentiation, viral infection, aging, yeast sporulation, and cancer development. Although the exact mechanisms connecting histone cleavage to gene expression are still emerging, it is clear that this process represents a novel epigenetic transcriptional mechanism intertwined with chromatin dynamics. This review explores known histone tail cleavage events, the proteolytic enzymes involved, their impact on gene expression, and future research directions in this evolving field.

An atlas of caspase cleavage events in differentiating muscle cells.

Executioner caspases, such as caspase-3, are known to induce apoptosis, but in other contexts, they can control very different fates, including cell differentiation and neuronal plasticity. While hundreds of caspase substrates are known to be specifically targeted during cell death, we know very little about how caspase activity brings about non-apoptotic fates. Here, we report the first proteome identification of cleavage events in C2C12 cells undergoing myogenic differentiation and its comparison to undifferentiated or dying C2C12 cells. These data have identified new caspase substrates, including caspase substrates specifically associated with differentiation, and show that caspases are regulating proteins involved in myogenesis in myotubes, several days after caspase-3 initiated differentiation. Cytoskeletal proteins emerged as a major group of non-apoptotic caspase substrates. We also identified proteins with well-established roles in muscle differentiation as substrates cleaved in differentiating cells.

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.

TermineR: Extracting information on endogenous proteolytic processing from shotgun proteomics data.

State-of-the-art mass spectrometers combined with modern bioinformatics algorithms for peptide-to-spectrum matching (PSM) with robust statistical scoring allow for more variable features (i.e., post-translational modifications) being reliably identified from (tandem-) mass spectrometry data, often without the need for biochemical enrichment. Semi-specific proteome searches, that enforce a theoretical enzymatic digestion to solely the N- or C-terminal end, allow to identify of native protein termini or those arising from endogenous proteolytic activity (also referred to as "neo-N-termini" analysis or "N-terminomics"). Nevertheless, deriving biological meaning from these search outputs can be challenging in terms of data mining and analysis. Thus, we introduce TermineR, a data analysis approach for the (1) annotation of peptides according to their enzymatic cleavage specificity and known protein processing features, (2) differential abundance and enrichment analysis of N-terminal sequence patterns, and (3) visualization of neo-N-termini location. We illustrate the use of TermineR by applying it to tandem mass tag (TMT)-based proteomics data of a mouse model of polycystic kidney disease, and assess the semi-specific searches for biological interpretation of cleavage events and the variable contribution of proteolytic products to general protein abundance. The TermineR approach and example data are available as an R package at https://github.com/MiguelCos/TermineR.

Efficacious human metapneumovirus vaccine based on AI-guided engineering of a closed prefusion trimer

The prefusion conformation of human metapneumovirus fusion protein (hMPV Pre-F) is critical for eliciting the most potent neutralizing antibodies and is the preferred immunogen for an efficacious vaccine against hMPV respiratory infections. Here we show that an additional cleavage event in the F protein allows closure and correct folding of the trimer. We therefore engineered the F protein to undergo double cleavage, which enabled screening for Pre-F stabilizing substitutions at the natively folded protomer interfaces. To identify these substitutions, we developed an AI convolutional classifier that successfully predicts complex polar interactions often overlooked by physics-based methods and visual inspection. The combination of additional processing, stabilization of interface regions and stabilization of the membrane-proximal stem, resulted in a Pre-F protein vaccine candidate without the need for a heterologous trimerization domain that exhibited high expression yields and thermostability. Cryo-EM analysis shows the complete ectodomain structure, including the stem, and a specific interaction of the newly identified cleaved C-terminus with the adjacent protomer. Importantly, the protein induces high and cross-neutralizing antibody responses resulting in near complete protection against hMPV challenge in cotton rats, making the highly stable, double-cleaved hMPV Pre-F trimer an attractive vaccine candidate.

SPRING licenses S1P-mediated cleavage of SREBP2 by displacing an inhibitory pro-domain

Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.

Cas12a domain flexibility guides R-loop formation and forces RuvC resetting

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Alternative proteoforms and proteoform-dependent assemblies in humans and plants

The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing goal in biology. Here, we systematically survey human and plant high-throughput bottom-up native proteomics data for protein truncation variants, where substantial regions of the full-length protein are missing from an observed protein product. In humans, Arabidopsis, and the green alga Chlamydomonas, approximately one percent of observed proteins show a short form, which we can assign by comparison to RNA isoforms as either likely deriving from transcript-directed processes or limited proteolysis. While some detected protein fragments align with known splice forms and protein cleavage events, multiple examples are previously undescribed, such as our observation of fibrocystin proteolysis and nuclear translocation in a green alga. We find that truncations occur almost entirely between structured protein domains, even when short forms are derived from transcript variants. Intriguingly, multiple endogenous protein truncations of phase-separating translational proteins resemble cleaved proteoforms produced by enteroviruses during infection. Some truncated proteins are also observed in both humans and plants, suggesting that they date to the last eukaryotic common ancestor. Finally, we describe novel proteoform-specific protein complexes, where the loss of a domain may accompany complex formation.

LGG-26. A ROLE FOR BRAF FUSION PARTNERS IN ONCOGENIC SIGNALING

Abstract t was originally hypothesized that loss of BRAF N-terminal negative regulatory domains, which are deleted in the KIAA1549:BRAF rearrangement, results in constitutive activation of the BRAF kinase. This model suggests that BRAF fusion partners are dispensable for BRAF oncogenic signaling. Paradoxically, our data suggest expression of truncated BRAF is insufficient for transformation and identify specific domains in KIAA1549 that are necessary for transformation. These domains are critical for regulating subcellular localization of the fusion protein and highlight an aberrant pattern of cellular localization that is not observed with wildtype BRAF. Altered subcellular localization results in proteolytic cleavage of KIAA1549:BRAF, and we identify the protease responsible for this cleavage event. This protease is therapeutically tractable and clinically relevant inhibitors have been developed, presenting a MAPK-independent mechanism to target the fusion. We also highlight an unexpected role for rare fusion partners, including FAM131B, in BRAF activation. In total, these data suggest that BRAF fusion partners are not indispensable for transformation as was previously thought, presenting unexplored opportunities to therapeutically target pLGG tumors with BRAF rearrangements. We are currently working on the IP measures required to disclose the specific enzyme(s) at ISPNO.

Natural-Target-Mimicking Translocation-Based Fluorescent Sensor for Detection of SARS-CoV-2 PLpro Protease Activity and Virus Infection in Living Cells.

Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1-4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins-red mScarlet I and green mNeonGreen-separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture.

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Inhibition of TBL1 cleavage alleviates doxorubicin-induced cardiomyocytes death by regulating the Wnt/β-catenin signal pathway.

Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with β-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/β-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.

UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

In vivo proteolytic profiling of the type I and type II metacaspases in Chlamydomonas reinhardtii exposed to salt stress.

Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using mass spectrometry-based degradomics approach, during control conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated the relevance of the predicted metacaspase substrates to the physiology of C. reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in the catabolism of carboxylic acids, while CrMCA-II plays an important role in photosynthesis and translation. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.