Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 in vivo.

Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts.

Myocardial ischemia-reperfusion (IR) (stunning) injury triggers changes in the proteome and degradome of the heart. Here, we utilize quantitative proteomics and comprehensive degradomics to investigate the molecular mechanisms of IR injury in isolated rat hearts. The control group underwent aerobic perfusion, while the IR injury group underwent 20 min of ischemia and 30 min of reperfusion to induce a stunning injury. As MMP-2 activation has been shown to contribute to myocardial injury, hearts also underwent IR injury with ARP-100, an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2 to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy, we quantified 4468 proteins in ventricular extracts, whereby 447 proteins showed significant alterations among the three groups. We then used subtiligase-mediated N-terminomic labeling to identify more than a hundred specific cleavage sites. Among these protease substrates, 15 were identified following IR injury. We identified alterations in numerous proteins involved in mitochondrial function and metabolism following IR injury. Our findings provide valuable insights into the biochemical mechanisms of myocardial IR injury, suggesting alterations in reactive oxygen/nitrogen species handling and generation, fatty acid metabolism, mitochondrial function and metabolism, and cardiomyocyte contraction.

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue.

Proteases are enzymes that induce irreversible post-translational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.

Gasdermin D activation in oligodendrocytes and microglia drives inflammatory demyelination in progressive multiple sclerosis.

Neuroinflammation coupled with demyelination and neuro-axonal damage in the central nervous system (CNS) contribute to disease advancement in progressive multiple sclerosis (P-MS). Inflammasome activation accompanied by proteolytic cleavage of gasdermin D (GSDMD) results in cellular hyperactivation and lytic death. Using multiple experimental platforms, we investigated the actions of GSDMD within the CNS and its contributions to P-MS. Brain tissues from persons with P-MS showed significantly increased expression of GSDMD, NINJ1, IL-1ß, and -18 within chronic active demyelinating lesions compared to MS normal appearing white matter and nonMS (control) white matter. Conditioned media (CM) from stimulated GSDMD+/+ human macrophages caused significantly greater cytotoxicity of oligodendroglial and neuronal cells, compared to CM from GSDMD-/- macrophages. Oligodendrocytes and CNS macrophages displayed increased Gsdmd immunoreactivity in the central corpus callosum (CCC) of cuprizone (CPZ)-exposed Gsdmd+/+ mice, associated with greater demyelination and reduced oligodendrocyte precursor cell proliferation, compared to CPZ-exposed Gsdmd-/- animals. CPZ-exposed Gsdmd+/+ mice exhibited significantly increased G-ratios and reduced axonal densities in the CCC compared to CPZ-exposed Gsdmd-/- mice. Proteomic analyses revealed increased brain complement C1q proteins and hexokinases in CPZ-exposed Gsdmd-/- animals. [18F]FDG PET imaging showed increased glucose metabolism in the hippocampus and whole brain with intact neurobehavioral performance in Gsdmd-/- animals after CPZ exposure. GSDMD activation in CNS macrophages and oligodendrocytes contributes to inflammatory demyelination and neuroaxonal injury, offering mechanistic and potential therapeutic insights into P-MS pathogenesis.

CaspSites: A Database and Web Application for Experimentally Observed Human Caspase Substrates Using N-Terminomics.

CaspSites is a free-to-use database and web application for experimentally observed human caspase substrates using N-terminomics. It can be accessed and used by all users at the web URL www.caspsites.org. CaspSites stores cleavage site information identified for human caspases 1-9 in lysates and apoptotic cells, collected from their corresponding published studies. The database can be queried, viewed, and exported using the search page of the web application. The main parameters offered are protein substrate, cleavage site (P4-P4') residues, and individual caspase data sets, which can be connected using OR, AND, or NOT logical operators for custom user-built queries. CaspSites will be regularly updated with new experimental findings for understudied caspases, providing researchers insight into the distinctive roles human caspases play in cellular processes by identifying their target proteins in relation to each other.

The roles of intracellular proteolysis in cardiac ischemia-reperfusion injury.

Ischemic heart disease remains a leading cause of human mortality worldwide. One form of ischemic heart disease is ischemia-reperfusion injury caused by the reintroduction of blood supply to ischemic cardiac muscle. The short and long-term damage that occurs due to ischemia-reperfusion injury is partly due to the proteolysis of diverse protein substrates inside and outside of cardiomyocytes. Ischemia-reperfusion activates several diverse intracellular proteases, including, but not limited to, matrix metalloproteinases, calpains, cathepsins, and caspases. This review will focus on the biological roles, intracellular localization, proteolytic targets, and inhibitors of these proteases in cardiomyocytes following ischemia-reperfusion injury. Recognition of the intracellular function of each of these proteases includes defining their activation, proteolytic targets, and their inhibitors during myocardial ischemia-reperfusion injury. This review is a step toward a better understanding of protease activation and involvement in ischemic heart disease and developing new therapeutic strategies for its treatment.

A conserved acetylation switch enables pharmacological control of tubby-like protein stability.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

Biochemical Tools for Tracking Proteolysis.

All living organisms depend on tightly regulated cellular networks to control biological functions. Proteolysis is an important irreversible post-translational modification that regulates most, if not all, cellular processes. Proteases are a large family of enzymes that perform hydrolysis of protein substrates, leading to protein activation or degradation. The 473 known and 90 putative human proteases are divided into 5 main mechanistic groups: metalloproteases, serine proteases, cysteine proteases, threonine proteases, and aspartic acid proteases. Proteases are fundamental to all biological systems, and when dysregulated they profoundly influence disease progression. Inhibiting proteases has led to effective therapies for viral infections, cardiovascular disorders, and blood coagulation just to name a few. Between 5 and 10% of all pharmaceutical targets are proteases, despite limited knowledge about their biological roles. More than 50% of all human proteases have no known substrates. We present here a comprehensive list of all current known human proteases. We also present current and novel biochemical tools to characterize protease functions in vitro, in vivo, and ex vivo. These tools make it achievable to define both beneficial and detrimental activities of proteases in health and disease.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.

Diverse, evolving conformer populations drive distinct phenotypes in frontotemporal lobar degeneration caused by the same MAPT-P301L mutation.

Tau protein accumulation is a common denominator of major dementias, but this process is inhomogeneous, even when triggered by the same germline mutation. We considered stochastic misfolding of human tau conformers followed by templated conversion of native monomers as an underlying mechanism and derived sensitive conformational assays to test this concept. Assessments of brains from aged TgTauP301L transgenic mice revealed a prodromal state and three distinct signatures for misfolded tau. Frontotemporal lobar degeneration (FTLD)-MAPT-P301L patients with different clinical phenotypes also displayed three signatures, two resembling those found in TgTauP301L mice. As physicochemical and cell bioassays confirmed diverse tau strains in the mouse and human brain series, we conclude that evolution of diverse tau conformers is intrinsic to the pathogenesis of this uni-allelic form of tauopathy. In turn, effective therapeutic interventions in FTLD will need to address evolving repertoires of misfolded tau species rather than singular, static molecular targets.

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast.

The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ∼20-60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ∼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage.

Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles.

Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

Structure-Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product.

Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues. To understand thiocillin's structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin's macrocyclic ring. We report the identification of eight unique compounds more potent than wild-type thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin's macrocyclic structure revealed a striking requirement for a low-entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high-potency binding.

UBC9-dependent association between calnexin and protein tyrosine phosphatase 1B (PTP1B) at the endoplasmic reticulum.

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.

Unraveling the mechanism of cell death induced by chemical fibrils.

We previously discovered a small-molecule inducer of cell death, named 1541, that noncovalently self-assembles into chemical fibrils ('chemi-fibrils') and activates procaspase-3 in vitro. We report here that 1541-induced cell death is caused by the fibrillar rather than the soluble form of the drug. A short hairpin RNA screen reveals that knockdown of genes involved in endocytosis, vesicle trafficking and lysosomal acidification causes partial 1541 resistance. We confirm the role of these pathways using pharmacological inhibitors. Microscopy shows that the fluorescent chemi-fibrils accumulate in punctae inside cells that partially colocalize with lysosomes. Notably, the chemi-fibrils bind and induce liposome leakage in vitro, suggesting they may do the same in cells. The chemi-fibrils induce extensive proteolysis including caspase substrates, yet modulatory profiling reveals that chemi-fibrils form a distinct class from existing inducers of cell death. The chemi-fibrils share similarities with proteinaceous fibrils and may provide insight into their mechanism of cellular toxicity.

The DegraBase: a database of proteolysis in healthy and apoptotic human cells.

Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the α-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique α-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (∼10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (∼25% of total), and finally, other endoproteolytic cuts (∼65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells.

Application of N-Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein in Vivo.

Alternative α- and ß-cleavage events in the cellular prion protein (PrPC) central region generate fragments with distinct biochemical features that affect prion disease pathogenesis, but the assignment of precise cleavage positions has proven challenging. Exploiting mouse transgenic models expressing wild-type (WT) PrPC and an octarepeat region mutant allele (S3) with increased ß-fragmentation, cleavage sites were defined using LC-MS/MS in conjunction with N-terminal enzymatic labeling and chemical in-gel acetylation. Our studies profile the net proteolytic repertoire of the adult brain, as deduced from defining hundreds of proteolytic events in other proteins, and position individual cleavage events in PrPC α- and ß-target areas imputed from earlier, lower resolution methods; these latter analyses established site heterogeneity, with six cleavage sites positioned in the ß-cleavage region of WT PrPC and nine positions for S3 PrPC. Regarding α-cleavage, aside from reported N-termini at His110 and Val111, we identified a total of five shorter fragments in the brain of both mice lines. We infer that aminopeptidase activity in the brain could contribute to the ragged N-termini observed around PrPC's α- and ß-cleavage sites, with this work providing a point of departure for further in vivo studies of brain proteases.