Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly.

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.

The biosynthetic origin of ribofuranose in bacterial polysaccharides.

Bacterial surface polysaccharides are assembled by glycosyltransferase enzymes that typically use sugar nucleotide or polyprenyl-monophosphosugar activated donors. Characterized representatives exist for many monosaccharides but neither the donor nor the corresponding glycosyltransferases have been definitively identified for ribofuranose residues found in some polysaccharides. Klebsiella pneumoniae O-antigen polysaccharides provided prototypes to identify dual-domain ribofuranosyltransferase proteins catalyzing a two-step reaction sequence. Phosphoribosyl-5-phospho-D-ribosyl-α-1-diphosphate serves as the donor for a glycan acceptor-specific phosphoribosyl transferase (gPRT), and a more promiscuous phosphoribosyl-phosphatase (PRP) then removes the residual 5'-phosphate. The 2.5-Å resolution crystal structure of a dual-domain ribofuranosyltransferase ortholog from Thermobacillus composti revealed a PRP domain that conserves many features of the phosphatase members of the haloacid dehalogenase family, and a gPRT domain that diverges substantially from all previously characterized phosphoribosyl transferases. The gPRT represents a new glycosyltransferase fold conserved in the most abundant ribofuranosyltransferase family.

The Amyloid Fibril-Forming ß-Sheet Regions of Amyloid ß and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

Lipopolysaccharide O-antigens-bacterial glycans made to measure.

Lipopolysaccharides are critical components of bacterial outer membranes. The more conserved lipid A part of the lipopolysaccharide molecule is a major element in the permeability barrier imposed by the outer membrane and offers a pathogen-associated molecular pattern recognized by innate immune systems. In contrast, the long-chain O-antigen polysaccharide (O-PS) shows remarkable structural diversity and fulfills a range of functions, depending on bacterial lifestyles. O-PS production is vital for the success of clinically important Gram-negative pathogens. The biological properties and functions of O-PSs are mostly independent of specific structures, but the size distribution of O-PS chains is particularly important in many contexts. Despite the vast O-PS chemical diversity, most are produced in bacterial cells by two assembly strategies, and the different mechanisms employed in these pathways to regulate chain-length distribution are emerging. Here, we review our current understanding of the mechanisms involved in regulating O-PS chain-length distribution and discuss their impact on microbial cell biology.

In Vitro Characterization of a Multidomain Glycosyltransferase Using Fluorescently Tagged Synthetic Acceptors.

In vitro assays using fluorescently tagged sugar residues can facilitate the characterization of glycosyltransferase function. Here we describe the use of in vitro assays to characterize the three glycosyltransferase modules of the protein designated WbbB from Klebsiella pneumoniae O12. This protein combines key activities necessary to synthesize the O antigenic polysaccharide portion of lipopolysaccharide. The specificities of the three glycosyltransferases were investigated in vitro, using purified proteins, the activated donor sugars (dTDP-Rha, UDP-GlcNAc and CMP-ß-Kdo) and synthetic acceptors terminating in either α1,3-linked Rha or ß1,4-linked GlcNAc. The reaction products were verified by mass spectrometry and nuclear magnetic resonance methods.

Short Total Synthesis of Ajoene.

We describe a short total synthesis of ajoene, a major biologically active constituent of garlic. The instability of allicin as the only other known alternative starting material has led to the development of a reliable procedure for the synthesis of ajoene from simple building blocks that is also suitable for upscale operations.

Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA.

Protein adsorption and assembly at interfaces provide a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air-water and oil-water interfaces. Densely packed, high modulus monolayers form at air-water interfaces, leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil-water interfaces, leading to the production of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates from a low areal density of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small molecule surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic-lipophilic balance (HLB number) calculated based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries experiments reveal intriguing reversible and nonreversible modes of mechanical deformation, respectively. The mechanical robustness of the microcapsules and the ability to engineer their shape and to design highly specific binding responses through protein engineering suggest that these microcapsules may be useful for biomedical applications.

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a ß-linked Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.

Protein-Based Hydrogels for Tissue Engineering.

The tunable mechanical and structural properties of protein-based hydrogels make them excellent scaffolds for tissue engineering and repair. Moreover, using protein-based components provides the option to insert sequences associated with promoting both cellular adhesion to the substrate and overall cell growth. Protein-based hydrogel components are appealing for their structural designability, specific biological functionality, and stimuli-responsiveness. Here we present highlights in the field of protein-based hydrogels for tissue engineering applications including design requirements, components, and gel types.

Protein design: Past, present, and future.

Building on the pioneering work of Ho and DeGrado (J Am Chem Soc 1987, 109, 6751-6758) in the late 1980s, protein design approaches have revealed many fundamental features of protein structure and stability. We are now in the era that the early work presaged - the design of new proteins with practical applications and uses. Here we briefly survey some past milestones in protein design, in addition to highlighting recent progress and future aspirations.

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation.

Many protein misfolding diseases, for example, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimer's disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

Novel insights into protein misfolding diseases revealed by ion mobility-mass spectrometry.

Amyloid disorders incorporate a wide range of human diseases arising from the failure of a specific peptide or protein to adopt, or remain in, its native functional conformational state. These pathological conditions, such as Parkinson's disease, Alzheimer's disease and Huntington's disease are highly debilitating, exact enormous costs on both individuals and society, and are predicted to increase in prevalence. Consequently, they form the focus of a topical and rich area of current scientific research. A major goal in attempts to understand and treat protein misfolding diseases is to define the structures and interactions of protein species intermediate between fully folded and aggregated, and extract a description of the aggregation process. This has proven a difficult task due to the inability of traditional structural biology approaches to analyze structurally heterogeneous systems. Continued developments in instrumentation and analytical approaches have seen ion mobility-mass spectrometry (IM-MS) emerge as a complementary approach for protein structure determination, and in some cases, a structural biology tool in its own right. IM-MS is well suited to the study of protein misfolding, and has already yielded significant structural information for selected amyloidogenic systems during the aggregation process. This review describes IM-MS for protein structure investigation, and provides a summary of current research highlighting how this methodology has unequivocally and unprecedentedly provided structural and mechanistic detail pertaining to the oligomerization of a variety of disease related proteins.

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ.

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.

Dissociation from the oligomeric state is the rate-limiting step in fibril formation by kappa-casein.

Amyloid fibrils are aggregated and precipitated forms of protein in which the protein exists in highly ordered, long, unbranching threadlike formations that are stable and resistant to degradation by proteases. Fibril formation is an ordered process that typically involves the unfolding of a protein to partially folded states that subsequently interact and aggregate through a nucleation-dependent mechanism. Here we report on studies investigating the molecular basis of the inherent propensity of the milk protein, kappa-casein, to form amyloid fibrils. Using reduced and carboxymethylated kappa-casein (RCMkappa-CN), we show that fibril formation is accompanied by a characteristic increase in thioflavin T fluorescence intensity, solution turbidity, and beta-sheet content of the protein. However, the lag phase of RCMkappa-CN fibril formation is independent of protein concentration, and the rate of fibril formation does not increase upon the addition of seeds (preformed fibrils). Therefore, its mechanism of fibril formation differs from the archetypal nucleation-dependent aggregation mechanism. By digestion with trypsin or proteinase K and identification by mass spectrometry, we have determined that the region from Tyr(25) to Lys(86) is incorporated into the core of the fibrils. We suggest that this region, which is predicted to be aggregation-prone, accounts for the amyloidogenic nature of kappa-casein. Based on these data, we propose that fibril formation by RCMkappa-CN occurs through a novel mechanism whereby the rate-limiting step is the dissociation of an amyloidogenic precursor from an oligomeric state rather than the formation of stable nuclei, as has been described for most other fibril-forming systems.

To ignore or explore: top-down modulation of novelty processing.

Abstract Attending to novelty is a critical element of human behavior and learning. Novel events can serve as task-irrelevant distracters or as potential sources of engagement by interesting or important aspects of one's environment. An optimally functioning brain should have the capacity to respond differentially to novel events depending on the circumstances in which they occur. In the present study, a subject-controlled variant of the visual novelty oddball paradigm was employed under two different conditions in which novel stimuli were characterized either as distracters from a main task or as potentially meaningful "invitations" to explore the environment. Differences in context, derived from varying the emphasis of task instructions, strongly modulated both the behavioral and electrophysiological response to novelty. This modulation was not observed for processing earlier than the P3 component. Subjects who encountered novel events that served as distracters limited the amount of attention and processing resources they appropriated. Remarkably, under this condition, there were no differences in overall P3 amplitude, late positive slow-wave activity, or viewing duration between rare novel and frequent standard events. In contrast, subjects who encountered novel events as potential opportunities to explore augmented the attention and processing resources directed toward these events (as reflected by a larger P3 amplitude, late positive slow-wave activity, and longer viewing durations). Our results suggest that the processing of novelty within the visual modality involves several stages, including: (1) the relatively automatic detection of unfamiliar, novel stimuli (indexed by the N2); (2) the voluntary allocation of resources determined by the broader context in which a novel event occurs (indexed by the P3); and (3) the sustained processing of novelty (indexed by late positive slow-wave activity). This study provides evidence of the brain's ability to generate differential responses to novel events according to the circumstances under which they are encountered. It also points to a greater degree of top-down modulation of the processing of novelty than has been previously emphasized. We suggest that less commonly studied variables, such as subject control, may provide additional insight into the different ways in which novelty is processed.

Site-directed mutations in the C-terminal extension of human alphaB-crystallin affect chaperone function and block amyloid fibril formation.

BACKGROUND: Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including alphaB-crystallin, play a role in the prevention of protein deposition. METHODOLOGY/PRINCIPAL FINDINGS: A series of site-directed mutants of the human molecular chaperone, alphaB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of alphaB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of alphaB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. CONCLUSIONS/SIGNIFICANCE: Together, our results highlight the important role of the C-terminal region of alphaB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify alphaB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.

Increased responsiveness to novelty is associated with successful cognitive aging.

The animal literature suggests that exposure to more complex, novel environments promotes neurogenesis and cognitive performance in older animals. Studies in humans indicate that participation in intellectually stimulating activities may serve as a buffer against mental decline and help to sustain cognitive abilities. Here, we show that across old adults, increased responsiveness to novel events (as measured by viewing duration and the size of the P3 event-related potential) is strongly linked to better performance on neuropsychological tests, especially those involving attention/executive functions. Cognitively high performing old adults generate a larger P3 response to visual stimuli than cognitively average performing adults. These results suggest that cognitively high performing adults successfully manage the task by appropriating more resources and that the increased size of their P3 component represents a beneficial compensatory mechanism rather than less efficient processing.

Age-related differences in attention to novelty among cognitively high performing adults.

Age-related differences in attention to novel events were studied in well-matched, cognitively high performing old, middle-aged and young subjects. Event-related potentials were recorded during a visual novelty oddball task in which subjects controlled viewing durations that served as a behavioral measure of attentional allocation. All age groups had a larger P3 amplitude and longer viewing duration to novel than to standard stimuli, with no age-related differences in the magnitude of these effects, indicating old individuals were as engaged by the processing of novelty as younger adults. Old subjects had a larger, more anteriorly distributed P3 component to novels and standards. The increased P3 amplitude differs from prior reports of a diminished P3 response with processes, including aging, that have a potentially deleterious impact on the brain. We hypothesise that cognitively high performing old individuals successfully manage the task by relying on additional neural resources and perhaps more effortful frontal activity than their younger counterparts.

Age-related differences in novelty and target processing among cognitively high performing adults.

Previous research on age-related changes in ERP components in response to novel and target stimuli has not carefully controlled for differences in level of cognitive status between age groups, which may have contributed to the common findings of increased P3 latency, decreased P3 amplitude, and altered P3 scalp distribution. Here, cognitively high-performing (top third based on published norms) old, middle-aged, and young adults matched for IQ, education, and gender participated in a novelty oddball paradigm. There were no age-associated differences in P3 latency. Older adults had a larger, more anteriorly distributed P3 amplitude to all stimulus types, even repetitive standards, suggesting they may rely on increased resources and effortful frontal activity to successfully process any kind of visual stimulus. However, after controlling for this non-specific age-related processing difference, the amplitude and scalp distribution of the P3 component to novel and target stimuli were comparable across age groups, indicating that for cognitively high functioning elders there may be no age-related differences specific to the processing of novel and target events as indexed by the P3 component.