Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

Widespread macromolecular interaction perturbations in human genetic disorders.

How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.

Proteome-scale discovery of protein degradation and stabilization effectors.

Targeted protein degradation and stabilization are promising therapeutic modalities because of their potency, versatility and their potential to expand the druggable target space1,2. However, only a few of the hundreds of E3 ligases and deubiquitinases in the human proteome have been harnessed for this purpose, which substantially limits the potential of the approach. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation3-5, but there are currently no methods that can identify such effector proteins in a scalable and unbiased manner. Here we established a synthetic proteome-scale platform to functionally identify human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. Our results reveal that the human proteome contains a large cache of effectors of protein stability. The approach further enabled us to comprehensively compare the activities of human E3 ligases and deubiquitinases, identify and characterize non-canonical protein degraders and stabilizers and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3 ligases cereblon and VHL. Our study provides a functional catalogue of stability effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for the discovery of new proximity-dependent protein modulators.

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.

Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex.

Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here, we show that FMRP regulates translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca's area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases, but not FMRP-deficient mice. Thus, alterations in FMRP posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

Postmitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal chromatin, transcriptome, and identity.

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide long-term histone replacement in postmitotic cells, including neurons. Beyond replenishment, histone variants also play active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover. Here, we uncover crucial roles for the histone H3 variant H3.3 in neuronal development. We find that newborn cortical excitatory neurons, which have only just completed replication-coupled deposition of canonical H3.1 and H3.2, substantially accumulate H3.3 immediately postmitosis. Codeletion of H3.3-encoding genes H3f3a and H3f3b from newly postmitotic neurons abrogates H3.3 accumulation, markedly alters the histone posttranslational modification landscape, and causes widespread disruptions to the establishment of the neuronal transcriptome. These changes coincide with developmental phenotypes in neuronal identities and axon projections. Thus, preexisting, replication-dependent histones are insufficient for establishing neuronal chromatin and transcriptome; de novo H3.3 is required. Stage-dependent deletion of H3f3a and H3f3b from 1) cycling neural progenitor cells, 2) neurons immediately postmitosis, or 3) several days later, reveals the first postmitotic days to be a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, codeletion of H3f3a and H3f3b does not lead to immediate H3.3 loss, but causes progressive H3.3 depletion over several months without widespread transcriptional disruptions or cellular phenotypes. Our study thus uncovers key developmental roles for de novo H3.3 in establishing neuronal chromatin, transcriptome, identity, and connectivity immediately postmitosis that are distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.

Chromatin remodeler Arid1a regulates subplate neuron identity and wiring of cortical connectivity.

Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.

TBR1 directly represses Fezf2 to control the laminar origin and development of the corticospinal tract.

The corticospinal (CS) tract is involved in controlling discrete voluntary skilled movements in mammals. The CS tract arises exclusively from layer (L) 5 projection neurons of the cerebral cortex, and its formation requires L5 activity of Fezf2 (Fezl, Zfp312). How this L5-specific pattern of Fezf2 expression and CS axonal connectivity is established with such remarkable fidelity had remained elusive. Here we show that the transcription factor TBR1 directly binds the Fezf2 locus and represses its activity in L6 corticothalamic projection neurons to restrict the origin of the CS tract to L5. In Tbr1 null mutants, CS axons ectopically originate from L6 neurons in a Fezf2-dependent manner. Consistently, misexpression of Tbr1 in L5 CS neurons suppresses Fezf2 expression and effectively abolishes the CS tract. Taken together, our findings show that TBR1 is a direct transcriptional repressor of Fezf2 and a negative regulator of CS tract formation that restricts the laminar origin of CS axons specifically to L5.

Structural basis for RNA binding and homo-oligomer formation by influenza B virus nucleoprotein.

Influenza virus nucleoprotein (NP) is the major component of the viral ribonucleoprotein complex, which is crucial for the transcription and replication of the viral genome. We have determined the crystal structure of influenza B virus NP to a resolution of 3.2 Å. Influenza B NP contains a head, a body domain, and a tail loop. The electropositive groove between the head and body domains of influenza B NP is crucial for RNA binding. This groove also contains an extended flexible charged loop (amino acids [aa] 125 to 149), and two lysine clusters at the first half of this loop were shown to be crucial for binding RNA. Influenza B virus NP forms a crystallographic homotetramer by inserting the tail loop into the body domain of the neighboring NP molecule. A deeply buried salt bridge between R472 and E395 and a hydrophobic cluster at F468 are the major driving forces for the insertion. The analysis of the influenza B virus NP structure and function and comparisons with influenza A virus NP provide insights into the mechanisms of action and underpin efforts to design inhibitors for this class of proteins.

Strategies for membrane interaction proteomics: no mass spectrometry required.

Membrane-bound proteins are one of the most important protein types in the cell, and are involved in many major cell processes and signaling pathways. Most proteins, including those at membranes, must interact with other proteins to form complexes, which are essential for their function(s). In this review, we describe some of the major non-mass spectrometry-based methods and technologies used for the investigation of intracellular membrane protein complexes including Tango, fluorescence/bioluminescence resonance energy transfer (F/BRET), luminescence-based mammalian interactome mapping (LUMIER), protein-fragment complementation assay (PCA), and membrane yeast two-hybrid assay (MYTH). We highlight the advantages and drawbacks of these methods, describe recent studies utilizing these methods, and discuss some of the major findings in the study of membrane protein-based cell pathways.

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities.

Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.

SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons.

Neocortical projection neurons exhibit layer-specific molecular profiles and axonal connections. Here we show that the molecular identities of early-born subplate and deep-layer neurons are not acquired solely during generation or shortly thereafter but undergo progressive postmitotic refinement mediated by SOX5. Fezf2 and Bcl11b, transiently expressed in all subtypes of newly postmigratory early-born neurons, are subsequently downregulated in layer 6 and subplate neurons, thereby establishing their layer 5-enriched postnatal patterns. In Sox5-null mice, this downregulation is disrupted, and layer 6 and subplate neurons maintain an immature differentiation state, abnormally expressing these genes postnatally. Consistent with this disruption, SOX5 binds and represses a conserved enhancer near Fezf2. The Sox5-null neocortex exhibits failed preplate partition and laminar inversion of early-born neurons, loss of layer 5 subcerebral axons, and misrouting of subplate and layer 6 corticothalamic axons to the hypothalamus. Thus, SOX5 postmitotically regulates the migration, postmigratory differentiation, and subcortical projections of subplate and deep-layer neurons.

Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80.

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.

Robust elimination of genome-damaged cells safeguards against brain somatic aneuploidy following Knl1 deletion.

The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.

The Functional Study of the N-Terminal Region of Influenza B Virus Nucleoprotein.

Influenza nucleoprotein (NP) is a major component of the ribonucleoprotein (vRNP) in influenza virus, which functions for the transcription and replication of viral genome. Compared to the nucleoprotein of influenza A (ANP), the N-terminal region of influenza B nucleoprotein (BNP) is much extended. By virus reconstitution, we found that the first 38 residues are essential for viral growth. We further illustrated the function of BNP by mini-genome reconstitution, fluorescence microscopy, electron microscopy, light scattering and gel shift. Results show that the N terminus is involved in the formation of both higher homo-oligomers of BNP and BNP-RNA complex.

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae.

Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein-protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.

Effects of humidity on foil and vial packaging to preserve glucose and lactate test strips for disaster readiness.

OBJECTIVE: Efficient emergency and disaster response is challenged by environmental conditions exceeding test reagent storage and operating specifications. We assessed the effectiveness of vial and foil packaging in preserving point-of-care (POC) glucose and lactate test strip performance in humid conditions. METHODS: Glucose and lactate test strips in both packaging were exposed to mean relative humidity of 97.0 ± 1.1% in an environmental chamber for up to 168 hours. At defined time points, stressed strips were removed and tested in pairs with unstressed strips using whole blood samples spiked to glucose concentrations of 60, 100, and 250 mg/dL (n = 20 paired measurements per level). A Wilcoxon signed rank test was used to compare stressed and unstressed test strip measurements. RESULTS: Stressed glucose and lactate test strip measurements differed significantly from unstressed strips, and were inconsistent between experimental trials. Median glucose paired difference was as high as 12.5 mg/dL at the high glucose test concentration. Median lactate bias was -0.2 mmol/L. Stressed strips from vial (3) and foil (7) packaging failed to produce results. CONCLUSIONS: Both packaging designs appeared to protect glucose and lactate test strips for at least 1 week of high humidity stress. Documented strip failures revealed the need for improved manufacturing process.

Short-Term Thermal-Humidity Shock Affects Point-of-Care Glucose Testing: Implications for Health Professionals and Patients.

The objective was to assess the effects of short-term (≤1 hour) static high temperature and humidity stresses on the performance of point-of-care (POC) glucose test strips and meters. Glucose meters are used by medical responders and patients in a variety of settings including hospitals, clinics, homes, and the field. Reagent test strips and instruments are potentially exposed to austere environmental conditions. Glucose test strips and meters were exposed to a mean relative humidity of 83.0% (SD = 8.0%) and temperature of 42°C (107.6°F, SD = 3.2) in a Tenney BTRC environmental chamber. Stressed and unstressed glucose reagent strips and meters were tested with spiked blood samples (n = 40 measurements per time point for each of 4 trials) after 15, 30, 45, and 60 minutes of exposure. Wilcoxon's signed rank test was applied to compare measurements test strip and meter measurements to isolate and characterize the magnitude of meter versus test strip effects individually. Stressed POC meters and test strips produced elevated glucose results, with stressed meter bias as high as 20 mg/dL (17.7% error), and stressed test strip bias as high as 13 mg/dL (12.2% error). The aggregate stress effect on meter and test strips yielded a positive bias as high as 33 mg/dL (30.1% error) after 15 minutes of exposure. Short-term exposure (15 minutes) to high temperature and humidity can significantly affect the performance of POC glucose test strips and meters, with measurement biases that potentially affect clinical decision making and patient safety.