Spatially resolved profiling of protein conformation and interactions by biocompatible chemical cross-linking in living cells.

Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells. Leveraging structure refinement with cross-linking restraints, we identify subcellular-specific conformation heterogeneity of PTEN, revealing dynamic differences in its dual specificity domains between the nucleus and cytoplasm. Furthermore, by discerning conformational disparities, we identify 83 cytoplasm-exclusive and 109 nucleus-exclusive PTEN-interacting proteins, each associated with distinct biological functions. Upon induction of ubiquitin-proteasome system stress, we observe dynamic alterations in PTEN assembly and its interacting partners during translocation. These changes, including the identification of components and interaction sites, are characterized using the SPACX approach. Notably, SPACX enables identification of unique interacting proteins specific to PTEN isoforms, including PTEN and PTEN-Long, through the determination of sequence-specific cross-linking interfaces. These findings underscore the potential of SPACX to elucidate the functional diversity of proteins within distinct subcellular sociology.

Mutations in glioblastoma proteins do not disrupt epitope presentation and recognition, maintaining a specific CD8 T cell immune response potential.

Antigen-specific cytotoxic CD8 T cells are extremely effective in controlling tumor growth and have been the focus of immunotherapy approaches. We leverage in silico tools to investigate whether the occurrence of mutations in proteins previously described as immunogenic and highly expressed by glioblastoma multiforme (GBM), such as Epidermal Growth Factor Receptor (EGFR), Isocitrate Dehydrogenase 1 (IDH1), Phosphatase and Tensin homolog (PTEN) and Tumor Protein 53 (TP53), may be contributing to the differential presentation of immunogenic epitopes. We recovered Class I MHC binding information from wild-type and mutated proteins using the Immune Epitope Database (IEDB). After that, we built peptide-MHC (pMHC-I) models in HLA-arena, followed by hierarchical clustering analysis based on electrostatic surface features from each complex. We identified point mutations that are determinants for the presentation of a set of peptides from TP53 protein. We point to structural features in the pMHC-I complexes of wild-type and mutated peptides, which may play a role in the recognition of CD8 T cells. To further explore these features, we performed 100 ns molecular dynamics simulations for the peptide pairs (wt/mut) selected. In pursuit of novel therapeutic targets for GBM treatment, we selected peptides where our predictive results indicated that mutations would not disrupt epitope presentation, thereby maintaining a specific CD8 T cell immune response. These peptides hold potential for future GBM interventions, including peptide-based or mRNA vaccine development applications.

Understanding P-Rex regulation: structural breakthroughs and emerging perspectives.

Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by ∼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gßγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis.

The NTE domain of PTENα/ß promotes cancer progression by interacting with WDR5 via its SSSRRSS motif.

PTENα/ß, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/ß to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/ß associated cancers. These findings not only shed light on the important role of the PTENα/ß-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.

Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

Comparative Protein Structural Network Analysis Reveals C-Terminal Tail Phosphorylation Structural Communication Fingerprint in PTEN-Associated Mutations in Autism and Cancer.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tightly regulated dual-specificity phosphatase and key regulator of the PI3K/AKT/mTOR signaling pathway. PTEN phosphorylation at its carboxy-terminal tail (CTT) serine/threonine cluster negatively regulates its tumor suppressor function by inducing a stable, closed, and inactive conformation. Germline PTEN mutations predispose individuals to PTEN hamartoma tumor syndrome (PHTS), a rare inherited cancer syndrome and, intriguingly, one of the most common causes of autism spectrum disorder (ASD). However, the mechanistic details that govern phosphorylated CTT catalytic conformational dynamics in the context of PHTS-associated mutations are unknown. Here, we utilized a comparative protein structure network (PSN)-based approach to investigate PTEN CTT phosphorylation-induced conformational dynamics specific to PTEN-ASD compared to PTEN-cancer phenotypes. Results from our study show differences in structural flexibility, inter-residue contacts, and allosteric communication patterns mediated by CTT phosphorylation, differentiating PTEN-ASD and PTEN-cancer phenotypes. Further, we identified perturbations among global metapaths and community network connections within the active site and inter-domain regions, indicating the significance of these regions in transmitting information across the PSN. Together, our studies provide a mechanistic underpinning of allosteric regulation through the coupled interplay of CTT phosphorylation conformational dynamics in PTEN-ASD and PTEN-cancer mutations. Importantly, the detailed atomistic interactions and structural consequences of PTEN variants reveal potential allosteric druggable target sites as a viable and currently unexplored treatment approach for individuals with different PHTS-associated mutations.

Structural and Dynamic Effects of PTEN C-Terminal Tail Phosphorylation.

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene encodes a tightly regulated dual-specificity phosphatase that serves as the master regulator of PI3K/AKT/mTOR signaling. The carboxy-terminal tail (CTT) is key to regulation and harbors multiple phosphorylation sites (Ser/Thr residues 380-385). CTT phosphorylation suppresses the phosphatase activity by inducing a stable, closed conformation. However, little is known about the mechanisms of phosphorylation-induced CTT-deactivation dynamics. Using explicit solvent microsecond molecular dynamics simulations, we show that CTT phosphorylation leads to a partially collapsed conformation, which alters the secondary structure of PTEN and induces long-range conformational rearrangements that encompass the active site. The active site rearrangements prevent localization of PTEN to the membrane, precluding lipid phosphatase activity. Notably, we have identified phosphorylation-induced allosteric coupling between the interdomain region and a hydrophobic site neighboring the active site in the phosphatase domain. Collectively, the results provide a mechanistic understanding of CTT phosphorylation dynamics and reveal potential druggable allosteric sites in a previously believed clinically undruggable protein.

Long noncoding RNA NEAT1 inhibits the acetylation of PTEN through the miR-524-5p /HDAC1 axis to promote the proliferation and invasion of laryngeal cancer cells.

Long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) is abnormally expressed in numerous tumors and functions as an oncogene, but the role of NEAT1 in laryngocarcinoma is largely unknown. Our study validated that NEAT1 expression was markedly upregulated in laryngocarcinoma tissues and cells. Downregulation of NEAT1 dramatically suppressed cell proliferation and invasion through inhibiting miR-524-5p expression. Additionally, NEAT1 overexpression promoted cell growth and metastasis, while overexpression of miR-524-5p could reverse the effect. NEAT1 increased the expression of histone deacetylase 1 gene (HDAC1) via sponging miR-524-5p. Mechanistically, overexpression of HDAC1 recovered the cancer-inhibiting effects of miR-524-5p mimic or NEAT1 silence by deacetylation of tensin homolog deleted on chromosome ten (PTEN) and inhibiting AKT signal pathway. Moreover, in vivo experiments indicated that silence of NEAT1 signally suppressed tumor growth. Taken together, knockdown of NEAT1 suppressed laryngocarcinoma cell growth and metastasis by miR-524-5p/HDAC1/PTEN/AKT signal pathway, which provided a potential therapeutic target for laryngocarcinoma.

The structural basis of PTEN regulation by multi-site phosphorylation.

Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.

PTENα is responsible for protection of brain against oxidative stress during aging.

Neural cells are continuously subjected to oxidative stress arising from electrochemical activity, and cellular protection systems can turn on the oxidative stress response to detect and alleviate adverse conditions. However, the function and mechanism of the protective systems are complicated and remain largely elusive. We report that PTENα, an isoform of the PTEN family, mediates defense signaling in response to oxidative stress during brain aging. We show that genetic ablation of Ptenα in mice increases oxidative stress and results in neuronal cell death, culminating in accelerated decline of cognition and motor coordination as age increases. PTENα maintains COX activity and promotes energy metabolism through abrogating NEDD4L-mediated degradation of COX4 in response to oxidative stress. In the presence of Parkinson's disease-associated mutation, PTENα loses the capability to protect COX4 and ameliorate defects caused by Ptenα deletion. Our study reveals an important role of PTENα in response to oxidative stress. We propose that dysregulation of PTENα signaling may accelerate the rate of brain aging and promote the development of neurodegenerative disorders.

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study.

C2 domains facilitate protein interactions with lipid bilayers in either a Ca2+-dependent or -independent manner. We used molecular dynamics (MD) simulations to explore six Ca2+-independent C2 domains, from KIBRA, PI3KC2α, RIM2, PTEN, SHIP2, and Smurf2. In coarse-grained MD simulations these C2 domains formed transient interactions with zwitterionic bilayers, compared with longer-lived interactions with anionic bilayers containing phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back, or side of the ß sandwich, whereas type II C2 domains bound canonically, via the top loops. C2 domains interacted strongly with membranes containing PIP2, causing bound anionic lipids to cluster around the protein. Binding modes were refined via atomistic simulations. For PTEN and SHIP2, CG simulations of their phosphatase plus C2 domains with PIP2-containing bilayers were also performed, and the roles of the two domains in membrane localization compared. These studies establish a simulation protocol for membrane-recognition proteins.

BGL3 inhibits papillary thyroid carcinoma progression via regulating PTEN stability.

PURPOSE: BGL3, a novel long non-coding RNA (lncRNA) that plays a crucial role in several human malignancies. However, the clinical significance and biological function of BGL3 in papillary thyroid carcinoma (PTC) have not been explored. Herein, we aimed to investigate the role of BGL3 in human PTC. METHODS: A total of 85 pairs of PTC and normal tissues were collected for clinicopathological analysis. Expression of BGL3 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of BGL3 on PTC cells ware determined by CCK-8, colony formation, EdU and wound healing assays. The molecular mechanism underlying BGL3 was tested by ChIP, Co-IP, RNA pull-down and luciferase reporter assays. In vivo experiments were conducted using xenografts in nude mice. RESULTS: BGL3 was significantly decreased in PTC tissues compared to adjacent normal thyroid tissues, and it was transcriptionally repressed by oncogene Myc. Low BGL3 is positively related to larger tumor size, lymph node metastasis, later TNM stage and poor prognosis. Overexpression of BGL3 inhibited PTC cell proliferation and migration in vitro, and reduced tumor size and lung metastasis nodules in vivo. BGL3 was mainly located in the cytoplasm, in which interacted with PTEN and recruited OTUD3, enhancing the de-ubiquitination effect of OTUD3 on PTEN, resulting in increasing PTEN protein stability and inactivating carcinogenic PI3K/AKT signaling. CONCLUSIONS: Our data underscore the critical tumor-inhibiting role of BGL3 in PTC via post-translational regulation of PTEN protein stability, which may serve as a novel therapeutic target and prognostic biomarker in human PTC.

Ubiquitin Ligase Activities of WWP1 Germline Variants K740N and N745S.

WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) N. Engl. J. Med.]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and in vitro enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.

Comprehensive in silico mutational-sensitivity analysis of PTEN establishes signature regions implicated in pathogenesis of Autism Spectrum Disorders.

An extensively studied cancer and Autism Spectrum Disorders (ASD) gene like PTEN provided an exclusive opportunity to map its mutational-landscape, compare and establish plausible genotypic predictors of ASD-associated phenotypic outcomes. Our exhaustive in silico analysis on 4252 SNPs using >30 tools identified increased mutational-density in exon7. Phosphatase domain, although evolutionarily conserved, had the most nsSNPs localised within signature regions. The evolutionarily variable C-terminal side contained the highest truncating-SNPs outside signature regions of C2 domain and most PTMs within C-tail site which displayed maximum intolerance to polymorphisms, and permitted benign but destabilising nsSNPs that enhanced its intrinsically-disordered nature. ASD-associated SNPs localised within ATP-binding motifs and Nuclear-Localising-Sequences were the most potent triggers of ASD manifestation. These, along with variations within P, WPD and TI loops, M1 within phosphatase domain, M2 and MoRFs of C2 domain, caused severe long-range conformational fluctuations altering PTEN's dynamic stability- not observed in variations outside signature regions. 3'UTR-SNPs affected 44 strong miRNA brain-specific targets; several 5' UTR-SNPs targeted transcription-factor POLR2A and 10 pathogenic Splice-Affecting-Variants were identified.

ACPS: An accurate bioinformatics tool for precision-based anti-cancer peptide generation via omics data.

The anti-cancer targets play a crucial role in the signaling processes of cells, and therefore, it becomes nearly impossible to engage these targets without affecting the native cellular function. Thus, an approach has been taken to develop an anti-cancer Scanner (ACPS) tool aimed toward the recognition of anti-cancer marks in the form of peptides. The proposed ACPS tool allows fast fingerprinting of the anti-cancer targets having extreme significance in the current bioinformatics research. There already exist some tools that offer these features on a single platform; however, the performance of ACPS was compared with the preexisting online tools and was observed that ACPS offers greater than 95% accuracy that is comparatively much higher. The anti-cancer marked sequences of proteins supplied by the operators are scanned against the anti-cancer target datasets via ACPS and provide precision-based anti-cancer peptides. The proposed tool has been contrived in PERL programming language, and this tool is the extended version of A-CaMP codes, which are highly scalable having an extensible application in cancer biology with robust coding architecture. The availability of tools like ACPS will greatly benefit researchers in the field of oncology and structure-based drug design.

Interactomic affinity profiling by holdup assay: Acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase.

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

Comprehensive characterization of amino acid positions in protein structures reveals molecular effect of missense variants.

Interpretation of the colossal number of genetic variants identified from sequencing applications is one of the major bottlenecks in clinical genetics, with the inference of the effect of amino acid-substituting missense variations on protein structure and function being especially challenging. Here we characterize the three-dimensional (3D) amino acid positions affected in pathogenic and population variants from 1,330 disease-associated genes using over 14,000 experimentally solved human protein structures. By measuring the statistical burden of variations (i.e., point mutations) from all genes on 40 3D protein features, accounting for the structural, chemical, and functional context of the variations' positions, we identify features that are generally associated with pathogenic and population missense variants. We then perform the same amino acid-level analysis individually for 24 protein functional classes, which reveals unique characteristics of the positions of the altered amino acids: We observe up to 46% divergence of the class-specific features from the general characteristics obtained by the analysis on all genes, which is consistent with the structural diversity of essential regions across different protein classes. We demonstrate that the function-specific 3D features of the variants match the readouts of mutagenesis experiments for BRCA1 and PTEN, and positively correlate with an independent set of clinically interpreted pathogenic and benign missense variants. Finally, we make our results available through a web server to foster accessibility and downstream research. Our findings represent a crucial step toward translational genetics, from highlighting the impact of mutations on protein structure to rationalizing the variants' pathogenicity in terms of the perturbed molecular mechanisms.

Rosetta-Enabled Structural Prediction of Permissive Loop Insertion Sites in Proteins.

While loop motifs frequently play a major role in protein function, our understanding of how to rationally engineer proteins with novel loop domains remains limited. In the absence of rational approaches, the incorporation of loop domains often destabilizes proteins, thereby requiring massive screening and selection to identify sites that can accommodate loop insertion. We developed a computational strategy for rapidly scanning the entire structure of a scaffold protein to determine the impact of loop insertion at all possible amino acid positions. This approach is based on the Rosetta kinematic loop modeling protocol and was demonstrated by identifying sites in lipase that were permissive to insertion of the LAP peptide. Interestingly, the identification of permissive sites was dependent on the contribution of the residues in the near-loop environment on the Rosetta score and did not correlate with conventional structural features (e.g., B-factors). As evidence of this, several insertion sites (e.g., following residues 17, 47-49, and 108), which were predicted and confirmed to be permissive, interrupted helices, while others (e.g., following residues 43, 67, 116, 119, and 121), which are situated in loop regions, were nonpermissive. This approach was further shown to be predictive for ß-glucosidase and human phosphatase and tensin homologue (PTEN), and to facilitate the engineering of insertion sites through in silico mutagenesis. By enabling the design of loop-containing protein libraries with high probabilities of soluble expression, this approach has broad implications in many areas of protein engineering, including antibody design, improving enzyme activity, and protein modification.

Parallel Chemoselective Profiling for Mapping Protein Structure.

Solution-based structural techniques complement high-resolution structural data by providing insight into the oft-missed links between protein structure and dynamics. Here, we present Parallel Chemoselective Profiling, a solution-based structural method for characterizing protein structure and dynamics. Our method utilizes deep mutational scanning saturation mutagenesis data to install amino acid residues with specific chemistries at defined positions on the solvent-exposed surface of a protein. Differences in the extent of labeling of installed mutant residues are quantified using targeted mass spectrometry, reporting on each residue's local environment and structural dynamics. Using our method, we studied how conformation-selective, ATP-competitive inhibitors affect the local and global structure and dynamics of full-length Src kinase. Our results highlight how parallel chemoselective profiling can be used to study a dynamic multi-domain protein, and suggest that our method will be a useful addition to the relatively small toolkit of existing protein footprinting techniques.

An Integrated Deep-Mutational-Scanning Approach Provides Clinical Insights on PTEN Genotype-Phenotype Relationships.

Germline variation in PTEN results in variable clinical presentations, including benign and malignant neoplasia and neurodevelopmental disorders. Despite decades of research, it remains unclear how the PTEN genotype is related to clinical outcomes. In this study, we combined two recent deep mutational scanning (DMS) datasets probing the effects of single amino acid variation on enzyme activity and steady-state cellular abundance with a large, well-curated clinical cohort of PTEN-variant carriers. We sought to connect variant-specific molecular phenotypes to the clinical outcomes of individuals with PTEN variants. We found that DMS data partially explain quantitative clinical traits, including head circumference and Cleveland Clinic (CC) score, which is a semiquantitative surrogate of disease burden. We built logistic regression models that use DMS and CADD scores to separate clinical PTEN variation from gnomAD control-only variation with high accuracy. By using a survival-like analysis, we identified molecular phenotype groups with differential risk of early cancer onset as well as lifetime risk of cancer. Finally, we identified classes of DMS-defined variants with significantly different risk levels for classical hamartoma-related features (odds ratio [OR] range of 4.1-102.9). In stark contrast, the risk for developing autism or developmental delay does not significantly change across variant classes (OR range of 5.4-12.4). Together, these findings highlight the potential impact of combining DMS datasets with rich clinical data and provide new insights that might guide personalized clinical decisions for PTEN-variant carriers.