Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.

Middle and Late Pleistocene Denisovan subsistence at Baishiya Karst Cave.

Genetic and fragmented palaeoanthropological data suggest that Denisovans were once widely distributed across eastern Eurasia1-3. Despite limited archaeological evidence, this indicates that Denisovans were capable of adapting to a highly diverse range of environments. Here we integrate zooarchaeological and proteomic analyses of the late Middle to Late Pleistocene faunal assemblage from Baishiya Karst Cave on the Tibetan Plateau, where a Denisovan mandible and Denisovan sedimentary mitochondrial DNA were found3,4. Using zooarchaeology by mass spectrometry, we identify a new hominin rib specimen that dates to approximately 48-32 thousand years ago (layer 3). Shotgun proteomic analysis taxonomically assigns this specimen to the Denisovan lineage, extending their presence at Baishiya Karst Cave well into the Late Pleistocene. Throughout the stratigraphic sequence, the faunal assemblage is dominated by Caprinae, together with megaherbivores, carnivores, small mammals and birds. The high proportion of anthropogenic modifications on the bone surfaces suggests that Denisovans were the primary agent of faunal accumulation. The chaîne opératoire of carcass processing indicates that animal taxa were exploited for their meat, marrow and hides, while bone was also used as raw material for the production of tools. Our results shed light on the behaviour of Denisovans and their adaptations to the diverse and fluctuating environments of the late Middle and Late Pleistocene of eastern Eurasia.

Homo sapiens reached the higher latitudes of Europe by 45,000 years ago.

The Middle to Upper Palaeolithic transition in Europe is associated with the regional disappearance of Neanderthals and the spread of Homo sapiens. Late Neanderthals persisted in western Europe several millennia after the occurrence of H. sapiens in eastern Europe1. Local hybridization between the two groups occurred2, but not on all occasions3. Archaeological evidence also indicates the presence of several technocomplexes during this transition, complicating our understanding and the association of behavioural adaptations with specific hominin groups4. One such technocomplex for which the makers are unknown is the Lincombian-Ranisian-Jerzmanowician (LRJ), which has been described in northwestern and central Europe5-8. Here we present the morphological and proteomic taxonomic identification, mitochondrial DNA analysis and direct radiocarbon dating of human remains directly associated with an LRJ assemblage at the site Ilsenhöhle in Ranis (Germany). These human remains are among the earliest directly dated Upper Palaeolithic H. sapiens remains in Eurasia. We show that early H. sapiens associated with the LRJ were present in central and northwestern Europe long before the extinction of late Neanderthals in southwestern Europe. Our results strengthen the notion of a patchwork of distinct human populations and technocomplexes present in Europe during this transitional period.

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.

Author Correction: The dental proteome of Homo antecessor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The dental proteome of Homo antecessor.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated1-5. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge6-8. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain)9,10 and Homo erectus from Dmanisi (Georgia)1, two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.

Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).

Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics.

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

Obtaining Complete Human Proteomes.

Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological context. In contrast to transcriptomics and genomics, the study of proteomes provides deeper insight into the dynamic regulatory layers encoded at the protein level, such as posttranslational modifications, subcellular localization, cell signaling, and protein-protein interactions. Currently, mass spectrometry (MS)-based proteomics is the technology of choice for studying proteomes at a system-wide scale, contributing to clinical biomarker discovery and fundamental molecular biology. MS technologies are continuously being developed to fulfill the requirements of speed, resolution, and quantitative accuracy, enabling the acquisition of comprehensive proteomes. In this review, we present how MS technology and acquisition methods have evolved to meet the requirements of cutting-edge proteomics research, which is describing the human proteome and its dynamic posttranslational modifications with unprecedented depth. Finally, we provide a perspective on studying proteomes at single-cell resolution.

Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.

Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression.

Dynamic lineage priming is driven via direct enhancer regulation by ERK.

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.

Enamel proteome shows that Gigantopithecus was an early diverging pongine.

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains1,3-6, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China7,8. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.

Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia.

BACKGROUND: Critical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal "cellular-cocktail" prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties. Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI. METHODS: Balb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach. RESULTS: AT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified. CONCLUSIONS: The combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.).

Quantitative proteome comparison of human hearts with those of model organisms.

Delineating human cardiac pathologies and their basic molecular mechanisms relies on research conducted in model organisms. Yet translating findings from preclinical models to humans present a significant challenge, in part due to differences in cardiac protein expression between humans and model organisms. Proteins immediately determine cellular function, yet their large-scale investigation in hearts has lagged behind those of genes and transcripts. Here, we set out to bridge this knowledge gap: By analyzing protein profiles in humans and commonly used model organisms across cardiac chambers, we determine their commonalities and regional differences. We analyzed cardiac tissue from each chamber of human, pig, horse, rat, mouse, and zebrafish in biological replicates. Using mass spectrometry-based proteomics workflows, we measured and evaluated the abundance of approximately 7,000 proteins in each species. The resulting knowledgebase of cardiac protein signatures is accessible through an online database: atlas.cardiacproteomics.com. Our combined analysis allows for quantitative evaluation of protein abundances across cardiac chambers, as well as comparisons of cardiac protein profiles across model organisms. Up to a quarter of proteins with differential abundances between atria and ventricles showed opposite chamber-specific enrichment between species; these included numerous proteins implicated in cardiac disease. The generated proteomics resource facilitates translational prospects of cardiac studies from model organisms to humans by comparisons of disease-linked protein networks across species.

A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase.

Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes.

THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1.

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair.

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.