Biology subjects

Kang, A.

Publications and source records attributed to Kang, A..

69 records · Page 4Linked to original sources

bioRxiv2022

Hallucination of closed repeat proteins containing central pockets

In pseudocyclic proteins such as TIM barrels, {beta} barrels, and some helical transmembrane channels, a single subunit is repeated in a cyclic pattern, giving rise to a central cavity which can serve as a pocket for ligand binding or enzymatic activity. Inspired by these proteins, we devised a deep learning-based approach to broadly exploring the space of closed repeat proteins starting from only a specification of the repeat number and length. Biophysical data for 38 structurally diverse pseudocyclic designs produced in E. coli are consistent with the design models, and two crystal structures we were able to obtain are very close to the designed structures. Docking studies suggest the diversity of folds and central pockets provide effective starting points for designing small molecule binders or enzymes.

molecular biology↗

bioRxiv2022

Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. Retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the novel nanoparticles we describe may be broadly useful in biotechnological applications.

biochemistry↗

bioRxiv2022

Hallucinating protein assemblies

Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here we use deep network hallucination to generate a wide range of symmetric protein homo-oligomers given only a specification of the number of protomers and the protomer length. Crystal structures of 7 designs are very close to the computational models (median RMSD: 0.6 [A]), as are 3 cryoEM structures of giant rings with up to 1550 residues, C33 symmetry, and 10 nanometer in diameter; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be created using deep learning, and pave the way for the design of increasingly complex nanomachines and biomaterials. One-sentence summaryDeep network-based protein design enables the generation of cyclic homo-oligomers across the nanoscopic scale.

biophysics↗

bioRxiv2022

Robust deep learning based protein sequence design using ProteinMPNN

While deep learning has revolutionized protein structure prediction, almost all experimentally characterized de novo protein designs have been generated using physically based approaches such as Rosetta. Here we describe a deep learning based protein sequence design method, ProteinMPNN, with outstanding performance in both in silico and experimental tests. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4%, compared to 32.9% for Rosetta. Incorporation of noise during training improves sequence recovery on protein structure models, and produces sequences which more robustly encode their structures as assessed using structure prediction algorithms. We demonstrate the broad utility and high accuracy of ProteinMPNN using X-ray crystallography, cryoEM and functional studies by rescuing previously failed designs, made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target binding proteins. One-sentence summaryA deep learning based protein sequence design method is described that is widely applicable to current design challenges and shows outstanding performance in both in silico and experimental tests.

biophysics↗

bioRxiv2021

Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, -helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

biochemistry↗

bioRxiv2021

Reconfigurable asymmetric protein assemblies through implicit negative design

Asymmetric multi-protein complexes that undergo subunit exchange play central roles in biology, but present a challenge for protein design. The individual components must contain interfaces enabling reversible addition to and dissociation from the complex, but be stable and well behaved in isolation. Here we employ a set of implicit negative design principles to generate beta sheet mediated heterodimers which enable the generation of a wide variety of structurally well defined asymmetric assemblies. Crystal structures of the heterodimers are very close to the design models, and unlike previously designed orthogonal heterodimer sets, the subunits are stable, folded and monomeric in isolation and rapidly assemble upon mixing. Rigid fusion of individual heterodimer halves to repeat proteins yields central assembly hubs that can bind two or three different proteins across different interfaces. We use these connectors to assemble linearly arranged hetero-oligomers with up to 6 unique components, branched hetero-oligomers, closed C4-symmetric two-component rings, and hetero-oligomers assembled on a cyclic homo-oligomeric central hub, and demonstrate such complexes can readily reconfigure through subunit exchange. Our approach provides a general route to designing asymmetric reconfigurable protein systems.

synthetic biology↗

bioRxiv2021

Single-dose respiratory mucosal delivery of next-generation viral-vectored COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2

The emerging SARS-CoV-2 variants of concern (VOC) increasingly threaten the effectiveness of current first-generation COVID-19 vaccines that are administered intramuscularly and are designed to only target the spike protein. There is thus a pressing need to develop next-generation vaccine strategies to provide more broad and long-lasting protection. By using adenoviral vectors (Ad) of human and chimpanzee origin, we developed Ad-vectored trivalent COVID-19 vaccines expressing Spike-1, Nucleocapsid and RdRp antigens and evaluated them following single-dose intramuscular or intranasal immunization in murine models. We show that respiratory mucosal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the three-arm immunity, consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells, and mucosal trained innate immunity. We further show that single-dose intranasal immunization provides robust protection against not only the ancestral strain of SARS-CoV-2, but also two emerging VOC, B.1.1.7 and B.1.351. Our findings indicate that single-dose respiratory mucosal delivery of an Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy against current and future VOC. This strategy has great potential to be used not only to boost first-generation vaccine-induced immunity but also to expand the breadth of protective T cell immunity at the respiratory mucosa.

immunology↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗

bioRxiv2020

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

A goal of de novo protein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric protein assemblies using these structures. We first connect designed helical repeat proteins (DHRs) to designed helical bundle proteins (HBs) to generate a large library of heterodimeric and homooligomeric building blocks; the latter have cyclic symmetries ranging from C2 to C6. All of the building blocks have repeat proteins with accessible termini, which we take advantage of in a second round of architecture guided rigid helical fusion (WORMS) to generate larger symmetric assemblies including C3 and C5 cyclic and D2 dihedral rings, a tetrahedral cage, and a 120 subunit icosahedral cage. Characterization of the structures by small angle x-ray scattering, x-ray crystallography, and cryo-electron microscopy demonstrates that the hierarchical design approach can accurately and robustly generate a wide range of macromolecular assemblies; with a diameter of 43nm, the icosahedral nanocage is the largest structurally validated designed cage to date. The computational methods and building block sets described here provide a very general route to new de novo designed symmetric protein nanomaterials.

biochemistry↗

bioRxiv2020

Only 40% of the world's forests are in good health

Many global environmental agendas, including halting biodiversity loss, reversing land degradation, and limiting climate change, depend upon retaining forests with high ecological integrity, yet the scale and degree of forest modification remains poorly quantified and mapped. By integrating data on observed and inferred human pressures and an index of lost connectivity, we generate the first globally-consistent, continuous index of forest condition as determined by degree of anthropogenic modification. Globally, only 17.4 million km2 of forest (40.5%) have high landscape level integrity (mostly found in Canada, Russia, the Amazon, Central Africa and New Guinea) and only 27% of this area is found in nationally-designated protected areas. Of the forest in protected areas, only 56% has high landscape level integrity. Ambitious policies that prioritize the retention of forest integrity, especially in the most intact areas, are now urgently needed alongside current efforts aimed at halting deforestation and restoring the integrity of forests globally.

ecology↗

bioRxiv2020

Immune reactivity to neurofilaments and dipeptide repeats in ALS progression

ObjectiveTo test antibody response and formation of immune-complexes to neurofilaments and dipeptide-repeats, the translational products of the mutated C9orf72 gene, as potential biomarkers for clinical stratification of amyotrophic lateral sclerosis (ALS). MethodsUsing neurofilament protein isoforms plasma expression as reference, antibodies and immune-complexes against neurofilament-light, medium and heavy chain and poly-(GP)-GR dipeptide-repeats were tested in blood from 105 fast and slow progressing ALS patients, 26 C9orf72 mutation carriers (C9+ve) ALS patients and 77 healthy controls (HC) using single-molecule and immune-capture assays. Longitudinal antibody/immune-complex responses were measured in serial blood samples from 37 (including 11 C9+ve) patients. ResultsAntibodies and immune-complex reactivity was higher in ALS patients than HC, particularly in C9+ve ALS patients, and modestly correlated with total neurofilament protein isoforms (r:0.24 p=0.002; r:0.18 p=0.02 respectively). Neurofilament-light immune-complexes and neurofilament-heavy antibodies had the best diagnostic performances distinguishing ALS subtypes from HC (AUC=0.68 p<0.01; AUC=0.68 p<0.001 respectively). Neurofilament-light immune-complexes (AUC=0.69 p<0.01) and poly-(GP) dipeptide-repeats antibodies (AUC=0.71 p<0.001) separated C9+ve from C9-ve patients. Multivariate mortality hazard ratio and Kaplan-Meier analyses showed low neurofilament-heavy antibody levels associated with increased survival. Longitudinal analysis identified raising levels of antibodies against neurofilaments in fast progressing ALS and of neurofilament-light immune-complexes in C9+ve patients. InterpretationC9+ve and fast progressing ALS patients have a distinct neurofilament and dipeptide-repeat immuno-phenotype, with increasing levels of blood neurofilament-light immune-complexes and neurofilament antibodies with disease progression. The study of the expression of these biomarkers in the natural history of ALS may shed light on disease initiation and progression and provide novel pharmacodynamic biomarkers in emerging C9orf72 gene silencing therapies.

neuroscience↗

bioRxiv2019

Rapamycin rejuvenates oral health in aging mice

Periodontal disease is an age-associated disorder clinically defined by periodontal bone loss, inflammation of the specialized tissues that surround and support the tooth, and microbiome dysbiosis. Currently, there is no therapy for reversing periodontal disease, and treatment is generally restricted to preventive measures or tooth extraction. The FDA-approved drug rapamycin slows aging and extends lifespan in multiple organisms, including mice. Here we demonstrate that short-term treatment with rapamycin rejuvenates the aged oral cavity of elderly mice, including regeneration of periodontal bone, attenuation of gingival and periodontal bone inflammation, and revertive shift of the oral microbiome toward a more youthful composition. This provides a geroscience strategy to potentially rejuvenate oral health and reverse periodontal disease in the elderly. Single Sentence SummaryShort-term treatment with rapamycin reverses periodontal bone loss, attenuates inflammation, and remodels the oral microbiome toward a more youthful state.

pathology↗

bioRxiv2019

The desmosomal cadherin Desmoglein-3 regulates YAP and phospho-YAP in keratinocyte responses to mechanical forces

Desmoglein 3 (Dsg3), plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction. A direct comparison was made with E-cadherin, a well-characterized mechanosensor, in human keratinocytes. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in expression and localization of junction assembly proteins. Knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of YAP, a mechanosensor and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 forms a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to both strain-or substrate rigidity-induced nuclear relocalization of YAP and phospho-YAP. We showed that PKP1 seemed to be the key in such a complex formation since its silencing resulted in Dsg3 disruption at the junctions with concomitant loss of pYAP in the peripheral cytoplasm. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes as well as cell proliferation in keratinocytes. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

cell biology↗

bioRxiv2019

Spontaneous isomerization of long-lived proteins provides a molecular mechanism for the lysosomal failure observed in Alzheimer’s disease

Proteinaceous aggregation is a well-known observable in Alzheimers disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely the cathepsins). For example, isomerization of aspartic acid into L-isoAsp prevents digestion of the N-terminal portion of A{beta} by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although A{beta} is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.

biochemistry↗

bioRxiv2018

The Pemphigus Vulgaris antigen desmoglein-3 suppresses p53 function via the YAP-Hippo pathway

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways implicated in the pathogenesis the PV blistering disease. We show here that expression of Dsg3 may directly influence p53, a key transcription factor governing the response to cellular stress. Dsg3 depletion caused increased p53 and apoptosis, an effect that was further enhanced by UV and mechanical strain and reversed by Dsg3 gain-of-function studies. Analysis in Dsg3-/- mouse skin confirmed increased p53/p21/caspase-3 compared to Dsg3+/- control in vivo. This Dsg3-p53 pathway involved YAP since Dsg3 forms a complex with YAP and regulates its expression and localization. Analysis of PV patient samples detected increased p53/YAP with diffuse cytoplasmic and/or nuclear staining in cells surrounding blisters. Treatment of keratinocytes with PV sera evoked pronounced p53/YAP expression. Collectively, our findings establish a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, suggesting that this pathway, involving YAP-Hippo control of skin homeostasis, is disrupted in PV.

cell biology↗