Chaperone BiP controls ER stress sensor Ire1 through interactions with its oligomers.

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

An AAGAB-to-CCDC32 handover mechanism controls the assembly of the AP2 adaptor complex.

Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and ß2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.

Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity.

The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders.

Type 1 fimbria and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family.

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.

Chemical chaperones to the rescue of Alport syndrome?

Alport syndrome is a hereditary kidney disease caused by collagen IV mutations that interfere with the formation and deposition of the α3α4α5 protomer into the glomerular basement membrane. In this issue, Yu et al. show that the chemical chaperone tauroursodeoxycholic acid prevented kidney structural changes and function decline in mice with a pathogenic missense Col4a3 mutation by increasing mutant α3α4α5 protomer glomerular basement membrane deposition and preventing podocyte apoptosis induced by endoplasmic reticulum stress.

Optogenetic induction of mechanical muscle stress identifies myosin regulatory ubiquitin ligase NHL-1 in C. elegans.

Mechanical stress during muscle contraction is a constant threat to proteome integrity. However, there is a lack of experimental systems to identify critical proteostasis regulators under mechanical stress conditions. Here, we present the transgenic Caenorhabditis elegans model OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant results in sustained muscle contraction and mechanical stress. Using OptIMMuS, combined with proximity labeling and mass spectrometry, we identify regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. One of these is the TRIM E3 ligase NHL-1, which interacts with UNC-45 and muscle myosin in genetic epistasis and co-immunoprecipitation experiments. We provide evidence that the ubiquitylation activity of NHL-1 regulates myosin levels and functionality under mechanical stress. In the future, OptIMMuS will help to identify muscle-specific proteostasis regulators of therapeutic relevance.

Structure and dimerization properties of the plant-specific copper chaperone CCH.

Copper chaperones of the ATX1 family are found in a wide range of organisms where these essential soluble carriers strictly control the transport of monovalent copper across the cytoplasm to various targets in diverse cellular compartments thereby preventing detrimental radical formation catalyzed by the free metal ion. Notably, the ATX1 family in plants contains two distinct forms of the cellular copper carrier. In addition to ATX1 having orthologs in other species, they also contain the copper chaperone CCH. The latter features an extra C-terminal extension whose function is still unknown. The secondary structure of this extension was predicted to be disordered in previous studies, although this has not been experimentally confirmed. Solution NMR studies on purified CCH presented in this study disclose that this region is intrinsically disordered regardless of the chaperone's copper loading state. Further biophysical analyses of the purified metallochaperone provide evidence that the C-terminal extension stabilizes chaperone dimerization in the copper-free and copper-bound states. A variant of CCH lacking the C-terminal extension, termed CCHΔ, shows weaker dimerization but similar copper binding. Computational studies further corroborate the stabilizing role of the C-terminal extension in chaperone dimerization and identify key residues that are vital to maintaining dimer stability.

The HtrA chaperone monitors sortase-assembled pilus biogenesis in Enterococcus faecalis.

Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the OG1RF ΔsrtAΔhtrA background partially restores the observed defects of the ΔsrtAΔhtrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal resistance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.

Pathogenic R163W Variant of the Copper Chaperone for Sod1 (Ccs) Functions as an Anti-chaperone.

The copper chaperone for Sod1 (Ccs) is a metallochaperone that plays a multifaceted role in the maturation of Cu,Zn superoxide dismutase (Sod1). The Ccs mutation R163W was identified in an infant with fatal neurological abnormalities. Based on a comprehensive structural and functional analysis, we developed the first data-driven model for R163W-related pathogenic phenotypes. The work here confirms previous findings that the substitution of arginine with tryptophan at this site, which is located adjacent to a conserved Zn binding site, creates an unstable Zn-deficient protein that loses its ability to efficiently activate Sod1. Intriguingly, R163W Ccs can reduce copper (i.e., Cu(II) → Cu(I)) bound in its Sod1-like domain (D2), and this novel redox event is accompanied by disulfide bond formation. The loss of Zn binding, along with the unusual ability to bind copper in D2, diverts R163W Ccs toward aggregation. The remarkably high affinity of D2 Cu(I) binding converts R163W from a Cu chaperone to a Cu scavenger that accelerates Sod1 deactivation (i.e., an Anti-chaperone). Overall, these findings present a first-of-its-kind molecular mechanism for Ccs dysfunction that leads to pathogenesis in humans.

Curcumin suppresses copper accumulation in non-small cell lung cancer by binding ATOX1.

BACKGROUND: Non-small cell lung cancer (NSCLC) is associated with intracellular copper accumulation. Antioxidant 1 (ATOX1) is a copper chaperone. This study aimed to analyze the anti-cancer effects of curcumin on the ATOX1-mediated copper pathway in NSCLC. METHODS: A binding activity between curcumin and ATOX1 was measured using molecular docking. NSCLC cells, A549 and H1299, were treated with different doses of curcumin (10, 20, 40 µM) or DC-AC50 (5, 10, 20 µM) for 24 h. The cell viability and levels of ATOX1, ATP7A and COX17 proteins were observed in cells. Overexpressing ATOX1 in cells was established by pcDNA3.1-ATOX1 transfection for 24 h. The ATOX1 overexpressing cells were treated with 40 µM curcumin or 20 µM DC-AC50 for 24 h to analyze the mechanism of curcumin in NSCLC treatment. Cell viability was measured by CCK-8, and levels of proteins were measured by western blotting. The copper level in cells was labeled by copper sensor-1. Moreover, nude mice models were induced by injection of A549 cells and treated with 20 mg/kg/d DC-AC50 or 40 mg/kg/d curcumin. Tumor growth was observed by measuring tumor volume and tumor weight. The levels of ATOX1, ATP7A and COX17 in tumors were measured by immunohistochemistry and western blotting. RESULTS: Curcumin bound to ATOX1 (score = -6.1 kcal/mol) and decreased the levels of ATOX1, ATP7A and COX17 proteins in NSCLC cells. The curcumin or DC-AC50 treatment suppressed cell viability by inhibiting the ATOX1-mediated copper signaling in NSCLC cells. The ATOX1 overexpression in cells significantly weakened the effects of curcumin on suppressing copper accumulation and the ATOX1-mediated copper pathway (p < 0.05). In mice models, curcumin or DC-AC50 treatment also suppressed tumor growth by suppressing the ATOX1-mediated copper pathway in tumors. CONCLUSION: This study demonstrated that curcumin bound ATOX1 to suppress copper accumulation in NSCLC cells, providing a new mechanism of curcumin for NSCLC treatment.

Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase.

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

Exoticin as a selective agonist of 6TM µ opioid receptors identifies endogenous chaperones essential for its activity.

BACKGROUND: Classical opioids are effective analgesics but carry various side effects, necessitating safer alternatives. Truncated six-transmembrane mu opioid receptors (6TM-µORs) mediate potent analgesia with fewer side effects and are a promising therapeutic target. However, few ligands known selectively target 6TM-µORs. Moreover, endogenous chaperones are believed essential for 6TM-µOR ligand binding and function. PURPOSE: To identify a 6TM-µOR selective agonist and elucidate requisite endogenous chaperones. METHODS: Virtual screening was used to identify promising selective 6TM-µOR agonists from traditional Chinese medicines. The role of 6TM-µOR in Exoticin analgesia was validated in loss- and gain-of-function models. APEX2 proteomics profiled proximal proteins under Exoticin or IBNtxA. Interactions were further characterized in vivo and in vitro. RESULTS: Exoticin was shortlisted for its selective binding to 6TM-µOR and ability to induce 6TM-µOR-dependent signal transduction. Exoticin analgesia was sensitive to ß-FNA and absent in E11 KO mice, but restored in mice infected with AAV-µOR1G. Slc3a2, Lrrc59, and Ppp1cb co-interacted with 6TM-µOR1G and were equally essential for Exoticin binding and 6TM-µOR1G activity. CONCLUSION: Exoticin is a promising selective agonist of 6TM µ opioid receptors with broad-spectrum analgesic efficacy but few side effects. Slc3a2, Lrrc59, Ppp1cb are endogenous chaperones essential for 6TM-µOR ligand binding and function.

The role of heat shock protein B8 in neuronal protection against oxidative stress and mitochondrial dysfunction: A literature review.

Excessive oxidative stress triggers cerebrovascular and neurodegenerative diseases resulting in acute and chronic brain injury. However, the underlying mechanisms remain unknown. Levels of small heat shock protein B8 (HSPB8), which is highly expressed in the brain, are known to be significantly elevated in cerebral injury models. Exogenous HSPB8 protects the brain against mitochondrial damage. One potential mechanism underlying this protection is that HSPB8 overexpression alleviates the mitochondria-dependent pathways of apoptosis; mitochondrial biogenesis, fission, and mitophagy. Overexpression of HSPB8 may therefore have potential as a clinical therapy for cerebrovascular and neurodegenerative diseases. This review provides an overview of advances in the protective effects of HSPB8 against excessive cerebral oxidative stress, including the modulation of mitochondrial dysfunction and potent signaling pathways.

Retinal Penetrating Adeno-Associated Virus.

Purpose: The most common method of delivery of genes to the outer retina uses recombinant adeno-associated virus (AAV) injected into the subretinal space using a surgical procedure. In contrast, most drugs are delivered to the retina using an intravitreal approach in an office setting. The objective of the current study was to develop AAV vectors that can reach the outer retina via intravitreal injection. Methods: Recently, we described a molecular chaperone (Nuc1) that enhanced the penetration of small and large molecules, including AAV, into the retina. The Nuc1 amino acid sequence or a truncated version of Nuc1 (IKV) was genetically incorporated into an exposed loop of AAV2/9 VP1 protein. These novel recombinant AAV vectors expressing green fluorescent protein (GFP) or nuclear factor erythroid 2 p45-related factor 2 (Nrf2) were injected into the vitreous of C57Bl/6J or Nrf2 knockout mice, respectively. The amount of GFP expression or oxidative stress as measured by 8-Hydroxy-2'-deoxyguanosine staining in C57Bl/6J or Nrf2 knockout mice, respectively, was quantified. Results: Incorporation of Nuc1 into AAV2/9 did not lead to significant expression of GFP in the murine retina. However, incorporation of IKV into AAV2/9 led to robust expression of GFP in photoreceptors and retinal pigment epithelium (RPE) via the intravitreal and subretinal routes of delivery. Furthermore, expression of Nrf2 using an IKV vector led to a reduction in oxidative stress in the retina of C57Bl/6J and Nrf2 knockout mice. Conclusions: We have developed a novel AAV vector that enables delivery of transgenes to the outer retina of mice, including photoreceptors and RPE following intravitreal injection.

POTRA domains of the TamA insertase interact with the outer membrane and modulate membrane properties.

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.

HSPB8 attenuates lipopolysaccharide­mediated acute lung injury in A549 cells by activating mitophagy.

Sepsis is a life­threatening multiple organ failure disease caused by an uncontrolled inflammatory response and can progress to acute lung injury (ALI). Heat­shock protein B8 (HSPB8) serves a cytoprotective role in multiple types of diseases; however, to the best of our knowledge, the regulatory role of HSPB8 in sepsis­induced ALI remains unclear. A549 human alveolar type II epithelial cells were treated with lipopolysaccharide (LPS) for 24 h to simulate a sepsis­induced ALI model. Cell transfection was performed to overexpress HSPB8, and cells were treated with mitochondrial division inhibitor­1 (Mdivi­1) for 2 h before LPS induction to assess the underlying mechanism. Protein expression was evaluated using western blotting and an immunofluorescence assay. Cytokines were examined using ELISA assay kits and antioxidant enzymes were examined using their detection kits. Cell apoptosis was detected using flow cytometry. The mitochondrial membrane potential was detected by JC­1 staining. HSPB8 was upregulated in A549 cells treated with LPS and HSPB8 overexpression attenuated LPS­induced inflammatory cytokine levels, oxidative stress and apoptosis in A549 cells. LPS inhibited mitophagy and reduced the mitochondrial membrane potential in A549 cells, which was partly inhibited by HSPB8 overexpression. Furthermore, Mdivi­1 decreased the inhibitory effect of HSPB8 on the inflammatory response, oxidative stress and apoptosis in LPS­treated A549 cells. In conclusion, HSPB8 overexpression attenuated the LPS­mediated inflammatory response, oxidative stress and apoptosis in A549 cells by promoting mitophagy, indicating HSPB8 as a potential therapeutic target in sepsis­induced ALI.

Association of RAN and RANBP2 Gene Polymorphisms With Glioma Susceptibility in Chinese Children.

BACKGROUND: Glioma is the most prevalent pediatric central nervous system malignancy. RAN, member RAS oncogene family (RAN), is a key signaling molecule that regulates the polymerization of microtubules during mitosis. RAN binding protein 2 (RANBP2) is involved in DNA replication, mitosis, metabolism, and tumorigenesis. The effects of RAN and RANBP2 gene polymorphisms on glioma susceptibility in Chinese children are currently unknown. AIMS: This study aimed to evaluate the association between RAN and RANBP2 gene polymorphisms and glioma susceptibility in Chinese children. METHODS AND RESULTS: We recruited 191 patients with glioma and 248 children without cancer for this case-control study. Polymerase chain reaction-based TaqMan was applied to gene sequencing and typing. Logistic regression model-calculated odds ratio and 95% confidence interval were used to verify whether the gene polymorphisms (RAN rs56109543 C>T, rs7132224 A>G, rs14035 C>T, and RANBP2 rs2462788 C>T) influence glioma susceptibility. Based on age, gender, tumor subtype, and clinical stage, stratified analyses of risk and protective genotypes were conducted. p values for mutant genotype analyses were all >0.05, indicating no significant correlation between these gene polymorphisms and glioma risk. CONCLUSION: RAN and RANBP2 gene polymorphisms were not found to be statistically significantly associated with glioma susceptibility in Chinese children. Other potential functional gene polymorphism loci of RAN and RANBP2 will need to be evaluated in the search for novel glioma biomarkers.

miR-15a targets the HSP90 co-chaperone Morgana in chronic myeloid leukemia.

Morgana is a ubiquitous HSP90 co-chaperone protein coded by the CHORDC1 gene. Morgana heterozygous mice develop with age a myeloid malignancy resembling human atypical myeloid leukemia (aCML), now renamed MDS/MPN with neutrophilia. Patients affected by this pathology exhibit low Morgana levels in the bone marrow (BM), suggesting that Morgana downregulation plays a causative role in the human malignancy. A decrease in Morgana expression levels is also evident in the BM of a subgroup of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients showing resistance or an incomplete response to imatinib. Despite the relevance of these data, the mechanism through which Morgana expression is downregulated in patients' bone marrow remains unclear. In this study, we investigated the possibility that Morgana expression is regulated by miRNAs and we demonstrated that Morgana is under the control of four miRNAs (miR-15a/b and miR-26a/b) and that miR-15a may account for Morgana downregulation in CML patients.

The Thioredoxin Fold Protein (TFP2) from Extreme Acidophilic Leptospirillum sp. CF-1 Is a Chaperedoxin-like Protein That Prevents the Aggregation of Proteins under Oxidative Stress.

Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

Substrate O-glycosylation actively regulates extracellular proteolysis.

Extracellular proteolysis critically regulates cellular and tissue responses and is often dysregulated in human diseases. The crosstalk between proteolytic processing and other major post-translational modifications (PTMs) is emerging as an important regulatory mechanism to modulate protease activity and maintain cellular and tissue homeostasis. Here, we focus on matrix metalloproteinase (MMP)-mediated cleavages and N-acetylgalactosamine (GalNAc)-type of O-glycosylation, two major PTMs of proteins in the extracellular space. We investigated the influence of truncated O-glycan trees, also referred to as Tn antigen, following the inactivation of C1GALT1-specific chaperone 1 (COSMC) on the general and MMP9-specific proteolytic processing in MDA-MB-231 breast cancer cells. Quantitative assessment of the proteome and N-terminome using terminal amine isotopic labelling of substrates (TAILS) technology revealed enhanced proteolysis by MMP9 within the extracellular proteomes of MDA-MB-231 cells expressing Tn antigen. In addition, we detected substantial modifications in the proteome and discovered novel ectodomain shedding events regulated by the truncation of O-glycans. These results highlight the critical role of mature O-glycosylation in fine-tuning proteolytic processing and proteome homeostasis by modulating protein susceptibility to proteolytic degradation. These data suggest a complex interplay between proteolysis and O-GalNAc glycosylation, possibly affecting cancer phenotypes.