Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease.

Mechanisms and regulation of substrate degradation by the 26S proteasome.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool.

Engineering novel adeno-associated viruses (AAVs) for improved delivery in the nervous system.

Harnessing adeno-associated virus (AAV) vectors for therapeutic gene delivery has emerged as a progressively promising strategy to treat disorders of both the central nervous system (CNS) and peripheral nervous system (PNS), and there are many ongoing clinical trials. However, unique physiological and molecular characteristics of the CNS and PNS pose obstacles to efficient vector delivery, ranging from the blood-brain barrier to the diverse nature of nervous system disorders. Engineering novel AAV capsids may help overcome these ongoing challenges and maximize therapeutic transgene delivery.

Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq.

Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators.

Genome Editing Therapy for the Blood: Success and Prospects.

Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer.

Exploring the sequence and structural determinants of the energy landscape from thermodynamically stable and kinetically trapped subtilisins: ISP1 and SbtE.

A protein's energy landscape, all the accessible conformations, their populations, and their dynamics of interconversion, is encoded in its primary sequence. While we have a good understanding of how a protein's primary sequence encodes its native state, we have a much weaker understanding of how sequence encodes the kinetic barriers such as unfolding and refolding. Here we have looked at two subtiliase homologs from the , Intracellular Subtilisin Protease 1 (ISP1) and Subtilisin E (SbtE) that are expected to have very different dynamics.

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA).

AI-based Prediction of Protein Corona Composition on DNA Nanostructures.

DNA nanotechnology has emerged as a powerful approach to engineering biophysical tools, therapeutics, and diagnostics because it enables the construction of designer nanoscale structures with high programmability. Based on DNA base pairing rules, nanostructure size, shape, surface functionality, and structural reconfiguration can be programmed with a degree of spatial, temporal, and energetic precision that is difficult to achieve with other methods. However, the properties and structure of DNA constructs are greatly altered due to spontaneous protein adsorption from biofluids.

Merlin phosphorylation regulates meningioma Wnt signaling and magnetic resonance imaging features.

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas.

Structural insight into synergistic activation of human 3-methylcrotonyl-CoA carboxylase.

The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far.

Animal and bacterial viruses share conserved mechanisms of immune evasion.

Animal and bacterial cells sense and defend against viral infections using evolutionarily conserved antiviral signaling pathways. Here, we show that viruses overcome host signaling using mechanisms of immune evasion that are directly shared across the eukaryotic and prokaryotic kingdoms of life. Structures of animal poxvirus proteins that inhibit host cGAS-STING signaling demonstrate architectural and catalytic active-site homology shared with bacteriophage Acb1 proteins, which inactivate CBASS anti-phage defense.

Birth of protein folds and functions in the virome.

The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts.

Oligomeric State and Drug Binding of the SARS-CoV-2 Envelope Protein Are Sensitive to the Ectodomain.

The envelope (E) protein of SARS-CoV-2 is the smallest of the three structural membrane proteins of the virus. E mediates budding of the progeny virus in the endoplasmic reticulum Golgi intermediate compartment of the cell. It also conducts ions, and this channel activity is associated with the pathogenicity of SARS-CoV-2.

Cryo-EM phase-plate images reveal unexpected levels of apparent specimen damage.

Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected.

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes.

Deep-learning-based design of synthetic orthologs of SH3 signaling domains.

Evolution-based deep generative models represent an exciting direction in understanding and designing proteins. An open question is whether such models can learn specialized functional constraints that control fitness in specific biological contexts. Here, we examine the ability of generative models to produce synthetic versions of Src-homology 3 (SH3) domains that mediate signaling in the Sho1 osmotic stress response pathway of yeast.

Temporal Resolution of Activity-Related Solvation Dynamics in the TIM Barrel Enzyme Murine Adenosine Deaminase.

Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein-solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent exposed helix-loop-helix moiety that presents the hydrophobic face of its long α-helix to the backside of bound substrate.

Activation of the helper NRC4 immune receptor forms a hexameric resistosome.

Innate immune responses to microbial pathogens are regulated by intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) in both the plant and animal kingdoms. Across plant innate immune systems, "helper" NLRs (hNLRs) work in coordination with "sensor" NLRs (sNLRs) to modulate disease resistance signaling pathways. Activation mechanisms of hNLRs based on structures are unknown.

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae.

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistronic genes, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for polycistronic expression in green algae.