Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency.

The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.

Novel computational biology modeling system can accurately forecast response to neoadjuvant therapy in early breast cancer.

BACKGROUND: Generalizable population-based studies are unable to account for individual tumor heterogeneity that contributes to variability in a patient's response to physician-chosen therapy. Although molecular characterization of tumors has advanced precision medicine, in early-stage and locally advanced breast cancer patients, predicting a patient's response to neoadjuvant therapy (NAT) remains a gap in current clinical practice. Here, we perform a study in an independent cohort of early-stage and locally advanced breast cancer patients to forecast tumor response to NAT and assess the stability of a previously validated biophysical simulation platform. METHODS: A single-blinded study was performed using a retrospective database from a single institution (9/2014-12/2020). Patients included: ≥ 18 years with breast cancer who completed NAT, with pre-treatment dynamic contrast enhanced magnetic resonance imaging. Demographics, chemotherapy, baseline (pre-treatment) MRI and pathologic data were input into the TumorScope Predict (TS) biophysical simulation platform to generate predictions. Primary outcomes included predictions of pathological complete response (pCR) versus residual disease (RD) and final volume for each tumor. For validation, post-NAT predicted pCR and tumor volumes were compared to actual pathological assessment and MRI-assessed volumes. Predicted pCR was pre-defined as residual tumor volume ≤ 0.01 cm3 (≥ 99.9% reduction). RESULTS: The cohort consisted of eighty patients; 36 Caucasian and 40 African American. Most tumors were high-grade (54.4% grade 3) invasive ductal carcinomas (90.0%). Receptor subtypes included hormone receptor positive (HR+)/human epidermal growth factor receptor 2 positive (HER2+, 30%), HR+/HER2- (35%), HR-/HER2+ (12.5%) and triple negative breast cancer (TNBC, 22.5%). Simulated tumor volume was significantly correlated with post-treatment radiographic MRI calculated volumes (r = 0.53, p = 1.3 × 10-7, mean absolute error of 6.57%). TS prediction of pCR compared favorably to pathological assessment (pCR: TS n = 28; Path n = 27; RD: TS n = 52; Path n = 53), for an overall accuracy of 91.2% (95% CI: 82.8% - 96.4%; Clopper-Pearson interval). Five-year risk of recurrence demonstrated similar prognostic performance between TS predictions (Hazard ratio (HR): - 1.99; 95% CI [- 3.96, - 0.02]; p = 0.043) and clinically assessed pCR (HR: - 1.76; 95% CI [- 3.75, 0.23]; p = 0.054). CONCLUSION: We demonstrated TS ability to simulate and model tumor in vivo conditions in silico and forecast volume response to NAT across breast tumor subtypes.

Magnesium transporter 1 (MAGT1) deficiency causes selective defects in N-linked glycosylation and expression of immune-response genes.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-of-function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg2+ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg2+ levels and that reduced Mg2+ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.

STAT5B: A Differential Regulator of the Life and Death of CD4+ Effector Memory T Cells.

Understanding the control of Ag restimulation-induced T cell death (RICD), especially in cancer immunotherapy, where highly proliferating T cells will encounter potentially large amounts of tumor Ags, is important now more than ever. It has been known that growth cytokines make T cells susceptible to RICD, but the precise molecular mediators that govern this in T cell subsets is unknown until now. STAT proteins are a family of transcription factors that regulate gene expression programs underlying key immunological processes. In particular, STAT5 is known to favor the generation and survival of memory T cells. In this study, we report an unexpected role for STAT5 signaling in the death of effector memory T (TEM) cells in mice and humans. TEM cell death was prevented with neutralizing anti-IL-2 Ab or STAT5/JAK3 inhibitors, indicating that STAT5 signaling drives RICD in TEM cells. Moreover, we identified a unique patient with a heterozygous missense mutation in the coiled-coil domain of STAT5B that presented with autoimmune lymphoproliferative syndrome-like features. Similar to Stat5b-/- mice, this patient exhibited increased CD4+ TEM cells in the peripheral blood. The mutant STAT5B protein dominantly interfered with STAT5-driven transcriptional activity, leading to global downregulation of STAT5-regulated genes in patient T cells upon IL-2 stimulation. Notably, CD4+ TEM cells from the patient were strikingly resistant to cell death by in vitro TCR restimulation, a finding that was recapitulated in Stat5b-/- mice. Hence, STAT5B is a crucial regulator of RICD in memory T cells in mice and humans.

Diagnostic accuracy of coronary CT angiography using 3rd-generation dual-source CT and automated tube voltage selection: Clinical application in a non-obese and obese patient population.

PURPOSE: To investigate diagnostic accuracy of 3rd-generation dual-source CT (DSCT) coronary angiography in obese and non-obese patients. METHODS: We retrospectively analyzed 76 patients who underwent coronary CT angiography (CCTA) and invasive coronary angiography. Prospectively ECG-triggered acquisition was performed with automated tube voltage selection (ATVS). Patients were dichotomized based on body mass index in groups A (<30 kg/m2, n = 37) and B (≥30 kg/m2, n = 39) and based on tube voltage in groups C (<120 kV, n = 46) and D (120 kV, n = 30). Coronary arteries were assessed for significant stenoses (≥50 % luminal narrowing) and diagnostic accuracy was calculated. RESULTS: Per-patient overall sensitivity, specificity, positive predictive value, negative predictive value (NPV) and accuracy were 96.9 %, 95.5 %, 93.9 %, 97.7 % and 96.1 %, respectively. Sensitivity and NPV were lower in groups B and D compared to groups A and C, but no statistically significant differences were observed (group A vs. B: sensitivity, 100.0 % vs. 93.3 %, p = 0.9493; NPV, 100 % vs. 95.5 %, p = 0.9812; group C vs. D: sensitivity, 100.0 % vs. 92.3 %, p = 0.8462; NPV, 100.0 % vs. 94.1 %, p = 0.8285). CONCLUSION: CCTA using 3rd-generation DSCT and (ATVS) provides high diagnostic accuracy in both non-obese and obese patients. KEY POINTS: • Coronary CTA provides high diagnostic accuracy in non-obese and obese patients. • Diagnostic accuracy between obese and non-obese patients showed no significant difference. • <120 kV studies were performed in 44 % of obese patients. • Current radiation dose-saving approaches can be applied independent of body habitus.

Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment.

BACKGROUND: Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. OBJECTIVE: We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. METHODS: Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. RESULTS: Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. CONCLUSIONS: Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.

Pan-Cancer, Genome-Scale Metabolic Network Analysis of over 10,000 Patients Elucidates Relationship between Metabolism and Survival.

Despite the high variability in cancer biology, cancers nevertheless exhibit cohesive hallmarks across multiple cancer types, notably dysregulated metabolism. Metabolism plays a central role in cancer biology, and shifts in metabolic pathways have been linked to tumor aggressiveness and likelihood of response to therapy. We therefore sought to interrogate metabolism across cancer types and understand how intrinsic modes of metabolism vary within and across indications and how they relate to patient prognosis. We used context specific genome-scale metabolic modeling to simulate metabolism across 10,915 patients from 34 cancer types from The Cancer Genome Atlas and the MMRF-COMMPASS study. We found that cancer metabolism clustered into modes characterized by differential glycolysis, oxidative phosphorylation, and growth rate. We also found that the simulated activities of metabolic pathways are intrinsically prognostic across cancer types, especially tumor growth rate, fatty acid biosynthesis, folate metabolism, oxidative phosphorylation, steroid metabolism, and glutathione metabolism. This work shows the prognostic power of individual patient metabolic modeling across multiple cancer types. Additionally, it shows that analyzing large-scale models of cancer metabolism with survival information provides unique insights into underlying relationships across cancer types and suggests how therapies designed for one cancer type may be repurposed for use in others.

Minimalist design of water-soluble cross-beta architecture.

Demonstrated successes of protein design and engineering suggest significant potential to produce diverse protein architectures and assemblies beyond those found in nature. Here, we describe a new class of synthetic protein architecture through the successful design and atomic structures of water-soluble cross-beta proteins. The cross-beta motif is formed from the lamination of successive beta-sheet layers, and it is abundantly observed in the core of insoluble amyloid fibrils associated with protein-misfolding diseases. Despite its prominence, cross-beta has been designed only in the context of insoluble aggregates of peptides or proteins. Cross-beta's recalcitrance to protein engineering and conspicuous absence among the known atomic structures of natural proteins thus makes it a challenging target for design in a water-soluble form. Through comparative analysis of the cross-beta structures of fibril-forming peptides, we identified rows of hydrophobic residues ("ladders") running across beta-strands of each beta-sheet layer as a minimal component of the cross-beta motif. Grafting a single ladder of hydrophobic residues designed from the Alzheimer's amyloid-beta peptide onto a large beta-sheet protein formed a dimeric protein with a cross-beta architecture that remained water-soluble, as revealed by solution analysis and x-ray crystal structures. These results demonstrate that the cross-beta motif is a stable architecture in water-soluble polypeptides and can be readily designed. Our results provide a new route for accessing the cross-beta structure and expanding the scope of protein design.

Next generation immuno-oncology tumor profiling using a rapid, non-invasive, computational biophysics biomarker in early-stage breast cancer.

Background: Immuno-oncology (IO) therapies targeting the PD-1/PD-L1 axis, such as immune checkpoint inhibitor (ICI) antibodies, have emerged as promising treatments for early-stage breast cancer (ESBC). Despite immunotherapy's clinical significance, the number of benefiting patients remains small, and the therapy can prompt severe immune-related events. Current pathologic and transcriptomic predictions of IO response are limited in terms of accuracy and rely on single-site biopsies, which cannot fully account for tumor heterogeneity. In addition, transcriptomic analyses are costly and time-consuming. We therefore constructed a computational biomarker coupling biophysical simulations and artificial intelligence-based tissue segmentation of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRIs), enabling IO response prediction across the entire tumor. Methods: By analyzing both single-cell and whole-tissue RNA-seq data from non-IO-treated ESBC patients, we associated gene expression levels of the PD-1/PD-L1 axis with local tumor biology. PD-L1 expression was then linked to biophysical features derived from DCE-MRIs to generate spatially- and temporally-resolved atlases (virtual tumors) of tumor biology, as well as the TumorIO biomarker of IO response. We quantified TumorIO within patient virtual tumors (n = 63) using integrative modeling to train and develop a corresponding TumorIO Score. Results: We validated the TumorIO biomarker and TumorIO Score in a small, independent cohort of IO-treated patients (n = 17) and correctly predicted pathologic complete response (pCR) in 15/17 individuals (88.2% accuracy), comprising 10/12 in triple negative breast cancer (TNBC) and 5/5 in HR+/HER2- tumors. We applied the TumorIO Score in a virtual clinical trial (n = 292) simulating ICI administration in an IO-naïve cohort that underwent standard chemotherapy. Using this approach, we predicted pCR rates of 67.1% for TNBC and 17.9% for HR+/HER2- tumors with addition of IO therapy; comparing favorably to empiric pCR rates derived from published trials utilizing ICI in both cancer subtypes. Conclusion: The TumorIO biomarker and TumorIO Score represent a next generation approach using integrative biophysical analysis to assess cancer responsiveness to immunotherapy. This computational biomarker performs as well as PD-L1 transcript levels in identifying a patient's likelihood of pCR following anti-PD-1 IO therapy. The TumorIO biomarker allows for rapid IO profiling of tumors and may confer high clinical decision impact to further enable personalized oncologic care.

A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the alpha-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an alpha-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

Molecular mechanism of Thioflavin-T binding to amyloid fibrils.

Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular probes. Since its first description in 1959, the fluorescent dye Thioflavin-T (ThT) has become among the most widely used "gold standards" for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clinical and basic science applications, the molecular mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, we review recent progress in the understanding of ThT-fibril interactions at an atomic resolution. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.

NF-κB Rel subunit exchange on a physiological timescale.

The Rel proteins of the NF-κB complex comprise one of the most investigated transcription factor families, forming a variety of hetero- or homodimers. Nevertheless, very little is known about the fundamental kinetics of NF-κB complex assembly, or the inter-conversion potential of dimerised Rel subunits. Here, we examined an unexplored aspect of NF-κB dynamics, focusing on the dissociation and reassociation of the canonical p50 and p65 Rel subunits and their ability to form new hetero- or homodimers. We employed a soluble expression system to enable the facile production of NF-κB Rel subunits, and verified these proteins display canonical NF-κB nucleic acid binding properties. Using a combination of biophysical techniques, we demonstrated that, at physiological temperatures, homodimeric Rel complexes routinely exchange subunits with a half-life of less than 10 min. In contrast, we found a dramatic preference for the formation of the p50/p65 heterodimer, which demonstrated a kinetic stability of at least an order of magnitude greater than either homodimer. These results suggest that specific DNA targets of either the p50 or p65 homodimers can only be targeted when these subunits are expressed exclusively, or with the intervention of additional post-translational modifications. Together, this work implies a new model of how cells can modulate NF-κB activity by fine-tuning the relative proportions of the p50 and p65 proteins, as well as their time of expression. This work thus provides a new quantitative interpretation of Rel dimer distribution in the cell, particularly for those who are developing mathematical models of NF-κB activity.

Monobodies as enabling tools for structural and mechanistic biology.

Monobodies, built with the scaffold of the fibronectin type III domain, are among the most well-established synthetic binding proteins. They promote crystallization of challenging molecular systems. They have strong tendency to bind to functional sites and thus serve as drug-like molecules that perturb the biological functions of their targets. Monobodies lack disulfide bonds and thus they are particularly suited as genetically encoded reagents to be used intracellularly. This article reviews recent monobody-enabled studies that reveal new structures, molecular mechanisms and potential therapeutic opportunities. A systematic analysis of the crystal structures of monobody-target complexes suggests important attributes that make monobodies effective crystallization chaperones.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Structures of single-layer ß-sheet proteins evolved from ß-hairpin repeats.

Free-standing single-layer ß-sheets are extremely rare in naturally occurring proteins, even though ß-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel ß-sheet proteins, comprised of three or four twisted ß-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of ß-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the ß-sheet. These new modular single-layer ß-sheet proteins may serve as a new model system for studying folding and design of ß-rich proteins.

Aromatic cluster mutations produce focal modulations of ß-sheet structure.

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model ß-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer ß-sheet of Borrelia outer surface protein A (OspA). This unusual ß-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the ß-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of ß-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA ß-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating ß-sheet conformation in protein design.

Heterozygous splice mutation in PIK3R1 causes human immunodeficiency with lymphoproliferation due to dominant activation of PI3K.

Class IA phosphatidylinositol 3-kinases (PI3K), which generate PIP3 as a signal for cell growth and proliferation, exist as an intracellular complex of a catalytic subunit bound to a regulatory subunit. We and others have previously reported that heterozygous mutations in PIK3CD encoding the p110δ catalytic PI3K subunit cause a unique disorder termed p110δ-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency (PASLI) disease. We report four patients from three families with a similar disease who harbor a recently reported heterozygous splice site mutation in PIK3R1, which encodes the p85α, p55α, and p50α regulatory PI3K subunits. These patients suffer from recurrent sinopulmonary infections and lymphoproliferation, exhibit hyperactive PI3K signaling, and have prominent expansion and skewing of peripheral blood CD8(+) T cells toward terminally differentiated senescent effector cells with short telomeres. The PIK3R1 splice site mutation causes skipping of an exon, corresponding to loss of amino acid residues 434-475 in the inter-SH2 domain. The mutant p85α protein is expressed at low levels in patient cells and activates PI3K signaling when overexpressed in T cells from healthy subjects due to qualitative and quantitative binding changes in the p85α-p110δ complex and failure of the C-terminal region to properly inhibit p110δ catalytic activity.

Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D.

The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg(2+)) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg(2+) causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8(+) T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg(2+) and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg(2+) in eukaryotic cells.

Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies.

A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic" (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of beta-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM beta-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the beta-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more beta-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.

Binding modes of thioflavin-T to the single-layer beta-sheet of the peptide self-assembly mimics.

Although the amyloid dye thioflavin-T (ThT) is among the most widely used tools in the study of amyloid fibrils, the mechanism by which ThT binds to fibrils and other beta-rich peptide self-assemblies remains elusive. The development of the water-soluble peptide self-assembly mimic (PSAM) system has provided a set of ideal model proteins for experimentally exploring the properties and minimal dye-binding requirements of amyloid fibrils. PSAMs consist of a single-layer beta-sheet (SLB) capped by two globular domains, which capture the flat, extended beta-sheet features common among fibril-like surfaces. Recently, a PSAM that binds to ThT with amyloid-like affinity (low micromolar K(d)) has been designed, and its crystal structure in the absence of bound ThT was determined. This PSAM thus provides a unique opportunity to examine the interactions of ThT with a beta-rich structure. Here, we present molecular dynamics simulations of the binding of ThT to this PSAM beta-sheet. We show that the primary binding site for ThT is along a shallow groove formed by adjacent Tyr and Leu residues on the beta-sheet surface. These simulations provide an atomic-scale rationale for this PSAM's experimentally determined dye-binding properties. Together, our results suggest that an aromatic-hydrophobic groove spanning across four consecutive beta-strands represents a minimal ThT binding site on amyloid fibrils. Grooves formed by aromatic-hydrophobic residues on amyloid fibril surfaces may therefore offer a generic mode of recognition for amyloid dyes.