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Over the last few years, a number of different protein assembly strategies have been developed, greatly expanding the toolbox for controlling macromolecular assembly. One of the most promising developments is a rapid protein ligation approach using a short polypeptide SpyTag and its partner, SpyCatcher derived from Streptococcus pyogenes fibronectin-binding protein, FbaB. To extend this technology, we have engineered and characterized a new Tag-Catcher pair from a related fibronectin-binding protein in Streptococcus dysgalactiae. The polypeptide Tag, named SdyTag, was constructed based on the native Cna protein B-type (CnaB) domain and was found to be highly unreactive to SpyCatcher. SpyCatcher has 320-fold specificity for its native SpyTag compared to SdyTag. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named SdyCatcherDANG short, compared to SpyCatcher. These Tag-Catcher pairs were used in combination to demonstrate specific sequential assembly of tagged proteins in vitro. We also demonstrated that the in vivo generation of circularized proteins in a Tag-Catcher specific manner where specific Tags can be left unreacted for use in subsequent ligation reactions. From the success of these experiments, we foresee the application of SdyTags and SpyTags, not only, for multiplexed control of protein assembly but also for the construction of novel protein architectures.

Mussels are opportunistic macrofouling organisms that can attach to most immersed solid surfaces, leading to serious economic and ecological consequences for the maritime and aquaculture industries. We demonstrate that lubricant-infused coatings exhibit very low preferential mussel attachment and ultralow adhesive strengths under both controlled laboratory conditions and in marine field studies. Detailed investigations across multiple length scales—from the molecular-scale characterization of deposited adhesive proteins to nanoscale contact mechanics to macroscale live observations—suggest that lubricant infusion considerably reduces fouling by deceiving the mechanosensing ability of mussels, deterring secretion of adhesive threads, and decreasing the molecular work of adhesion. Our study demonstrates that lubricant infusion represents an effective strategy to mitigate marine biofouling and provides insights into the physical mechanisms underlying adhesion prevention.

The squid beak displays a 200-fold stiffness gradient across its length. A battery of experiments, including ‘omics analysis and rheological measurements, now identifies two protein families that infiltrate and cross-link a porous chitin network to generate variable stiffness. The beak of the jumbo squid Dosidicus gigas is a fascinating example of how seamlessly nature builds with mechanically mismatched materials. A 200-fold stiffness gradient begins in the hydrated chitin of the soft beak base and gradually increases to maximum stiffness in the dehydrated distal rostrum. Here, we combined RNA-Seq and proteomics to show that the beak contains two protein families. One family consists of chitin-binding proteins (DgCBPs) that physically join chitin chains, whereas the other family comprises highly modular histidine-rich proteins (DgHBPs). We propose that DgHBPs play multiple key roles during beak bioprocessing, first by forming concentrated coacervate solutions that diffuse into the DgCBP-chitin scaffold, and second by inducing crosslinking via an abundant GHG sequence motif. These processes generate spatially controlled desolvation, resulting in the impressive biomechanical gradient. Our findings provide novel molecular-scale strategies for designing functional gradient materials.

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known ( ACY3 , HAAO, HNF1A , MAP3K11 ) and previously unidentified ( ABCD3 , CDKN2AIP , USH1C , VIL1 ) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1 , GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation. Here, the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells, and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant.

The Rbfox family of developmentally important splicing factors controls alternative splicing in a tissue-specific manner. Genome-wide analyses now show that more than half of Rbfox-binding sites are located distally from exons, that these distal sites are conserved and functionally important, and that long-range RNA-RNA secondary structures mediate distal splicing regulation by Rbfox. Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A +/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived β-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients. Heterozygous HNF1A mutations can give rise to maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. Here the authors show that MODY3-related HNF1A mutation in patient hiPSCderived pancreatic cells decreases glucose transporter GLUT2 expression due to compromised DNA binding.

Biomineralization, the process by which mineralized tissues grow and harden via biogenic mineral deposition, is a relatively lengthy process in many mineral-producing organisms, resulting in challenges to study the growth and biomineralization of complex hard mineralized tissues. Arthropods are ideal model organisms to study biomineralization because they regularly molt their exoskeletons and grow new ones in a relatively fast timescale, providing opportunities to track mineralization of entire tissues. Here, we monitored the biomineralization of the mantis shrimp dactyl club—a model bioapatite-based mineralized structure with exceptional mechanical properties—immediately after ecdysis until the formation of the fully functional club and unveil an unusual development mechanism. A flexible membrane initially folded within the club cavity expands to form the new club’s envelope. Mineralization proceeds inwards by mineral deposition from this membrane, which contains proteins regulating mineralization. Building a transcriptome of the club tissue and probing it with proteomic data, we identified and sequenced Club Mineralization Protein 1 (CMP-1), an abundant mildly phosphorylated protein from the flexible membrane suggested to be involved in calcium phosphate mineralization of the club, as indicated by in vitro studies using recombinant CMP-1. This work provides a comprehensive picture of the development of a complex hard tissue, from the secretion of its organic macromolecular template to the formation of the fully functional club.

Malassezia form the dominant eukaryotic microbial community on the human skin. The Malassezia genus possesses a repertoire of secretory hydrolytic enzymes involved in protein and lipid metabolism which alter the external cutaneous environment. The exact role of most Malassezia secreted enzymes, including those in interaction with the epithelial surface, is not well characterized. In this study, we compared the expression level of secreted proteases, lipases, phospholipases, and sphingomyelinases of Malassezia globosa in healthy subjects and seborrheic dermatitis or atopic dermatitis patients. We observed upregulated gene expression of the previously characterized secretory aspartyl protease MGSAP1 in both diseased groups, in lesional and non-lesional skin sites, as compared to healthy subjects. To explore the functional roles of MGSAP1 in skin disease, we generated a knockout mutant of the homologous protease MFSAP1 in the genetically tractable Malassezia furfur. We observed the loss of MFSAP1 resulted in dramatic changes in the cell adhesion and dispersal in both culture and a human 3D reconstituted epidermis model. In a murine model of Malassezia colonization, we further demonstrated Mfsap1 contributes to inflammation as observed by reduced edema and inflammatory cell infiltration with the knockout mutant versus wildtype. Taken together, we show that this dominant secretory Malassezia aspartyl protease has an important role in enabling a planktonic cellular state that can potentially aid in colonization and additionally as a virulence factor in barrier-compromised skin, further highlighting the importance of considering the contextual relevance when evaluating the functions of secreted microbial enzymes.

Large collections of annotated single-cell RNA sequencing (scRNA-seq) experiments are being generated across different organs, conditions and organisms on different platforms. The diversity, volume and complexity of this aggregated data requires new analysis techniques to extract actionable knowledge. Fundamental to most analysis are key abilities such as: identification of similar cells across different experiments and transferring annotations from an annotated dataset to an unannotated one. There have been many strategies explored in achieving these goals, and they focuses primarily on aligning and re-clustering datasets of interest. In this work, we are interested in exploring the applicability of deep metric learning methods as a form of distance function to capture similarity between cells and facilitate the transfer of cell type annotation for similar cells across different experiments. Toward this aim, we developed MapCell, a few-shot training approach using Siamese Neural Networks (SNNs) to learn a generalizable distance metric that can differentiate between single cell types. Requiring only a small training set, we demonstrated that SNN derived distance metric can perform accurate transfer of annotation across different scRNA-seq platforms, batches, species and also aid in flagging novel cell types.

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3′ untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism. Abnormal functions of RNA-binding proteins TAF15, FUS and TDP43 are associated with amyotrophic lateral sclerosis. Here, Kapeli et al . characterize the RNA targets of TAF15 and identify points of convergence and divergence between the targets of TAF15, FUS and TDP43 in several neuronal systems.