Amyloid ␤-protein (A␤) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of A␤ in AD is new evidence that oligomeric forms of A␤ are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of A␤, A␤42, with the disease. Detailed knowledge of the structure and assembly dynamics of A␤ thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that A␤ oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, sizeexclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of A␤40 and A␤42. Carefully prepared aggregate-free A␤40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, A␤42 preferentially formed pentamer͞hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to A␤40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that A␤42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of A␤42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.A myloid ␤-protein (A␤) fibril formation and deposition long have been linked to the neuropathogenesis of Alzheimer's disease (AD) (1-5). However, recent data have shown that oligomeric A␤ assembly intermediates are potent neurotoxins, and that these intermediates may be the key effectors of neurotoxicity in AD (6). In transgenic mice expressing the human amyloid ␤-protein precursor (A␤PP) and A␤, neurologic deficits develop before and independently of the appearance of amyloid deposits (6, 7). Importantly, soluble oligomeric forms of A␤ are neurotoxic in vitro (8-15) and in vivo (15). The main alloforms of A␤ found in amyloid deposits are 40 and 42 amino acids long (designated A␤40 and A␤42, respectively). Despite the small structural difference between these two peptides, they display distinct clinical, biological, and biophysical behavior. The concentration of secreted A␤42 is Ϸ10% that of A␤40, yet the longer form is the predominant component in parenchymal plaques (16)(17)(18)). An increase in the A␤42͞A␤40 concentration ratio is associated with familial forms of early onset AD (19,20). Treatments that reduce A␤42 levels have been shown to correlate with decreased risk for AD (21). In addition, A␤42 displays enhanced neurotoxicity relative to A␤40 (22-24). Studies of the kinetics of A␤ fibril formation have shown that A␤42 forms fibrils significantly faster than A␤40 (25), leading to the oftrepeated statement ''A␤42 is mor...

Assembly of monomeric amyloid ␤-protein (A␤) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight A␤40 and A␤42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight A␤40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight A␤42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant A␤ alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in A␤42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus selfassociation. A␤40 oligomerization was particularly sensitive to substitutions of Glu 22 Past studies of the role of the amyloid ␤-protein (A␤) 1 in Alzheimer's disease (AD) have linked the neurotoxicity of A␤ with its tendency to form amyloid fibrils (1-4). However, accumulating evidence suggests that soluble A␤ oligomers and prefibrillar aggregates are the proximal effectors of neurotoxicity in early stages of AD (for reviews, see Refs. 5-7). Soluble oligomeric forms of A␤, including dimers and trimers (8, 9), A␤-derived diffusible ligands (10 -16), and protofibrils (17-20) are neurotoxic. Transgenic mice expressing the human amyloid ␤-protein precursor (A␤PP) exhibit neurologic deficits prior to the appearance of amyloid deposits (21-23). In humans, A␤ oligomers have been detected in cerebrospinal fluid of Alzheimer's patients but not in age-matched controls (24). An A␤PP mutation causing reduction of total A␤ levels accompanied by accelerated A␤ assembly into protofibrils has been linked to early onset familial Alzheimer's disease (FAD) in a Swedish kindred (25). These findings support an important role for A␤ oligomers in the etiology of AD and thus make A␤ oligomers attractive therapeutic targets.If effective drug design strategies targeting A␤ oligomers are to be developed, detailed knowledge of the structures and assembly dynamics of these oligomers must be obtained. However, the biophysical and structural characterization of oligomeric A␤ assemblies has been difficult due to their metastable nature. Previously, we demonstrated that the size distribution of A␤ oligomers could be determined quantitatively (26, 27) using the technique photo-induced cross-linking of unmodified protei...

Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid -protein (A ) forms neurotoxic oligomers. The predominant in vivo A alloforms, A 40 and A 42, have distinct oligomerization pathways. A 42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. A 40 does not form these paranuclei, a fact which may explain the particularly strong linkage of A 42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met 35 is associated with altered A assembly, we examined the role of Met 35 in controlling A oligomerization. Oxidation of Met 35 in A 42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by A 40. Systematic structural alterations of the Cγ 35 -substituent group revealed that its electronic nature, rather than its size (van der Waals volume), was the factor controlling oligomerization pathway choice. Preventing assembly of toxic A 42 paranuclei through selective oxidation of Met 35 thus represents a potential therapeutic approach for AD.

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo–designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CART cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ-and CD28-containing endodomain signaling chain. We tested STOP-CART cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CART cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CART cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies. T cells engineered with CARs, hybrid molecules linking antigen binding to T-cell signaling endodomains, have mediated potent and durable responses against both chronic and acute B cell leukemias 1-4. CART cell therapy, however, is frequently associated with lifethreatening side effects, including cytokine release syndrome (CRS) and neurotoxicity. The clinical development of CART cells against solid tumors has proven challenging, but there is great optimism that next-generation CART cells will bring benefit to a broader range of cancer patients 5. Indeed, it is now well understood that physical and immunometabolic barriers upregulated in the solid tumor microenvironment as well as prolonged exposure to antigens can impair T-cell function and drive T-cell exhaustion 6. Innovative engineering strategies, such as the expression of cytokines, chemokines, decoy molecules or stimulatory ligands, are being developed to overcome these obstacles and have shown favorable preclinical responses 6-8. Safety, however, remains an important barrier to clinical entry because most solid tumor antigens targeted to date are also found in healthy tissues, sometimes leading to serious on-target off-tumor toxicity 9. The ability to control CART cell activity on command could greatly accelerate the clinical development of cellular immunotherapies. The above considerations have driven the development of CART cell control and safety systems 5 , such as drug-inducible suicide switches 10,11 , coinhibitory receptor (i)CARs that upon engagement with specific antigens suppress effector function 12 and split-signaling CART cells that require co-engagement of two ligands for full T-cell activation 13. SUPRA (split, universal and programmable) CARs 14 and a variety of universal CARs 15,16 have been developed that require administration of an adaptor protein to link nontumor antigen binding CART cells to tumor cells. While these approaches offer the possibility to sequentially target different tumor antigens (for ...

The use of major histocompatibility complex (MHC) tetramers in the detection and analysis of antigen-specific T cells has become more widespread since its introduction 11 years ago. Early challenges in the application of tetramer staining to CD4+ T cells centred around difficulties in the expression of various class II MHC allelic variants and the detection of low-frequency T cells in mixed populations. As many of the technical obstacles to class II MHC tetramer staining have been overcome, the focus has returned to uncertainties concerning how oligomer valency and T-cell receptor/MHC affinity affect tetramer binding. Such issues have become more important with an increase in the number of studies relying on direct ex vivo analysis of antigen-specific CD4+ T cells. In this review we discuss which problems in class II MHC tetramer staining have been solved to date, and which matters remain to be considered

Understanding the structural and assembly dynamics of the amyloid ␤-protein (A␤) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the A␤ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform "scanning PICUP." Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for A␤40) or 42 (for A␤42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil 3 ␣/␤ 3 ␤ transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr 1 -substituted homologues of A␤40 and A␤42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr 1 ]A␤40 fibrils. Substitution of A␤40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both A␤40 and A␤42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr 1 ) to alter A␤ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that A␤ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.Alzheimer disease (AD) 4 is the most common cause of latelife dementia (1) and is estimated to afflict more than 27 million people worldwide (2). An important etiologic hypothesis is that amyloid ␤-protein (A␤) oligomers are the proximate neurotoxins in AD. Substantial in vivo and in vitro evidence supports this hypothesis (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Neurotoxicity studies have shown that A␤ assemblies are potent neurotoxins (5,(13)(14)(15)(16)(17)(18)(19)(20), and the toxicity of some oligomers can be greater than that of the corresponding fibrils (21). Soluble A␤ oligomers inhibit hippocampal long term potentiation (4,5,13,15,17,18,22) and disrupt cognitive function (23). Compounds that bind and disrupt the formation of oligomers have been shown to block the neurotoxicity of A␤ (24,25). Importantly, recent studies in higher vertebrates (dogs) have shown that substantial reduction in amyloid deposits in the absence of decreases in oligomer concentration has little effect on recovery of neurological function (26).Recent studies of A␤ oligomers have sought to correlate oligomer size and biological activity. Oligomers in the supernatants of fibril preparations centrifuged at 100,000 ϫ g caused sustained calcium influx in rat hippocampal neurons, leading to calpain activati...

Background:The human equilibrative glucose transporter GLUT1 catalyzes accelerated-exchange transport, but the related isoform GLUT4 does not. Results: Substitution of GLUT1 transmembrane helix 6 into GLUT4 permits GLUT4 to catalyze accelerated-exchange, whereas GLUT4 helix 6 prevents accelerated-exchange in GLUT1. Conclusion: Transmembrane helix 6 sequence determines accelerated-exchange behavior. Significance: Transmembrane helices external to the transport pathway influence transport behavior.