An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.     

Experience-dependent emergence of a grouping bias

Humans share with non-human animals perceptual biases that might form the basis of complex cognitive abilities. One example comes from the principles described by the iambic–trochaic law (ITL). According to the ITL, sequences of sounds varying in duration are grouped as iambs, whereas sequences varying in intensity are grouped as trochees. These grouping biases have gained much attention because they might help pre-lexical infants bootstrap syntactic parameters (such as word order) in their language. Here, we explore how experience triggers the emergence of perceptual grouping biases in a non-human species. We familiarized rats with either long–short or short–long tone pairs. We then trained the animals to discriminate between sequences of alternating and randomly ordered tones. Results showed animals developed a grouping bias coherent with the exposure they had. Together with results observed in human adults and infants, these results suggest that experience modulates perceptual organizing principles that are present across species.     

The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.     

Pat1 contributes to the RNA binding activity of the Lsm1-7–Pat1 complex

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5′-to-3′ exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7–Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7–Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7–Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7–Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7–Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7–Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.     

Mechanistic insights into the inhibition of the CRISPR-Cas surveillance complex by anti-CRISPR protein AcrIF13

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide prokaryotes with nucleic acid–based adaptive immunity against infections of mobile genetic elements, including phages. To counteract this immune process, phages have evolved various anti-CRISPR (Acr) proteins which deactivate CRISPR-Cas–based immunity. However, the mechanisms of many of these Acr-mediated inhibitions are not clear. Here, we report the crystal structure of AcrIF13 and explore its inhibition mechanism. The structure of AcrIF13 is unique and displays a negatively charged surface. Additionally, biochemical studies identified that AcrIF13 interacts with the type I-F CRISPR-Cas surveillance complex (Csy complex) to block target DNA recognition and that the Cas5f-8f tail and Cas7.6f subunit of the Csy complex are specific binding targets of AcrIF13. Further mutational studies demonstrated that several negatively charged residues of AcrIF13 and positively charged residues of Cas8f and Cas7f of the Csy complex are involved in AcrIF13–Csy binding. Together, our findings provide mechanistic insights into the inhibition mechanism of AcrIF13 and further suggest the prevalence of the function of Acr proteins as DNA mimics.     

Interactomes of SARS‐CoV‐2 and human coronaviruses reveal host factors potentially affecting pathogenesis

Host–virus protein–protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity‐labeling strategies and identified 437 human proteins as the high‐confidence interacting proteins. Further characterization of these interactions and comparison to other large‐scale study of cellular responses to SARS‐CoV‐2 infection elucidated how distinct SARS‐CoV‐2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID‐19. The interactomes of two key SARS‐CoV‐2‐encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein–protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS‐CoV‐2 provides valuable resources for the understanding and treating of this disease.     

The role of self–other distinction in understanding others' mental and emotional states: neurocognitive mechanisms in children and adults

Social interactions come with the fundamental problem of trying to understand others'' mental and affective states while under the overpowering influence of one''s own concurrent thoughts and feelings. The ability to distinguish between simultaneous representations of others'' current experiences as well as our own is crucial to navigate our complex social environments successfully. The developmental building blocks of this ability and how this is given rise to by functional and structural brain development remains poorly understood. In this review, I outline some of the key findings on the role of self–other distinction in understanding others'' mental as well as emotional states in children and adults. I will begin by clarifying the crucial role for self–other distinction in avoiding egocentric attributions of one''s own cognitive as well as affective states to others in adults and outline the underlying neural circuitry in overcoming such egocentricity. This will provide the basis for a discussion of the emergence of self–other distinction in early childhood as well as developmental changes therein throughout childhood and into adulthood. I will demonstrate that self–other distinction of cognitive and emotional states is already dissociable early in development. Concomitantly, I will show that processes of self–other distinction in cognitive and affective domains rely on adjacent but distinct neural circuitry each with unique connectivity profiles, presumably related to the nature of the distinction that needs to be made.     

Structuring policy problems for plastics,the environment and human health: reflections from the UK

How can we strengthen the science–policy interface for plastics, the environment and human health? In a complex policy area with multiple stakeholders, it is important to clarify the nature of the particular plastics-related issue before trying to understand how to reconcile the supply and demand for evidence in policy. This article proposes a simple problem typology to assess the fundamental characteristics of a policy issue and thus identify appropriate processes for science–policy interactions. This is illustrated with two case studies from one UK Government Department, showing how policy and science meet over the environmental problems of plastics waste in the marine environment and on land. A problem-structuring methodology helps us understand why some policy issues can be addressed through relatively linear flows of science from experts to policymakers but why others demand a more reflexive approach to brokering the knowledge between science and policy. Suggestions are given at the end of the article for practical actions that can be taken on both sides.     

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex,but Not gH/gL,Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128–131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128–131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128–131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)₂ homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0–6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4–8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128–131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.     

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1–P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix–turn–helix domain (amino acids I13–R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1–D12) did not bind. The N-terminal M1–P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1–M234) and Tnp26 M1–P56 fragment. These findings indicate that the helix–turn–helix and disordered domains of Tnp26 play a role in Tnp26–TIR complex formation. Both domains are conserved in all members of the IS26 family.     

Bardet–Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self‐tolerance

Bardet–Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS‐induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.     

When phenology matters: age–size truncation alters population response to trophic mismatch

Climate-induced shifts in the timing of life-history events are a worldwide phenomenon, and these shifts can de-synchronize species interactions such as predator–prey relationships. In order to understand the ecological implications of altered seasonality, we need to consider how shifts in phenology interact with other agents of environmental change such as exploitation and disease spread, which commonly act to erode the demographic structure of wild populations. Using long-term observational data on the phenology and dynamics of a model predator–prey system (fish and zooplankton in Windermere, UK), we show that age–size truncation of the predator population alters the consequences of phenological mismatch for offspring survival and population abundance. Specifically, age–size truncation reduces intraspecific density regulation due to competition and cannibalism, and thereby amplifies the population sensitivity to climate-induced predator–prey asynchrony, which increases variability in predator abundance. High population variability poses major ecological and economic challenges as it can diminish sustainable harvest rates and increase the risk of population collapse. Our results stress the importance of maintaining within-population age–size diversity in order to buffer populations against phenological asynchrony, and highlight the need to consider interactive effects of environmental impacts if we are to understand and project complex ecological outcomes.     

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2–ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2–ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2–ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.     

Activation of Phosphatidylinositol 3-Kinase (PI3K) and Mitogen-activated Protein Kinase (MAPK) Signaling and the Consequent Induction of Transformation by Overexpressed 14-3-3γ Protein Require Specific Amino Acids within 14-3-3γ N-terminal Variable Region II

Members of the 14-3-3 superfamily regulate numerous cellular functions by binding phosphoproteins. The seven human isoforms (and the myriad of other eukaryotic 14-3-3 proteins) are highly conserved in amino acid sequence and secondary structure, yet there is abundant evidence that the various isoforms manifest disparate as well as common functions. Several of the human 14-3-3 isoforms are dysregulated in certain cancers and thus have been implicated in oncogenesis; experimentally, 14-3-3γ behaves as an oncogene, whereas 14-3-3σ acts as a tumor suppressor. In this study, we sought to localize these opposing phenotypes to specific regions of the two isoforms and then to individual amino acids therein. Using a bioinformatics approach, six variable regions (VRI–VRVI) were identified. Using this information, two sets of constructs were created in which N-terminal portions (including either VRI–IV or only VRI and VRII) of 14-3-3γ and 14-3-3σ were swapped; NIH3T3 cells overexpressing the four chimeric proteins were tested for transformation activity (focus formation, growth in soft agar) and activation of PI3K and MAPK signaling. We found that the specific phenotypes of 14-3-3γ are associated with the N-terminal 40 amino acids (VRI and VRII); in like fashion, VRI and VRII of 14-3-3σ dictated its tumor suppressor function. Using individual amino acid substitutions within the 14-3-3γ VRII, we identified two residues required for and two contributing to the γ-specific phenotypes. Our observations suggest that isoform-specific phenotypes are dictated by a relatively few amino acids within variable regions.     

Aboveground competition influences density‐dependent effects of cordgrass on sediment biogeochemistry

Interspecific interactions between plants influence plant phenotype, distribution, abundance, and community structure. Each of these can, in turn, impact sediment biogeochemistry. Although the population and community level impacts of these interactions have been extensively studied, less is known about their effect on sediment biogeochemistry. This is surprising given that many plants are categorized as foundation species that exert strong control on community structure. In southern California salt marshes, we used clipping experiments to manipulate aboveground neighbor presence to study interactions between two dominant plants, Pacific cordgrass (Spartina foliosa) and perennial pickleweed (Sarcocornia pacifica). We also measured how changes in cordgrass stem density influenced sediment biogeochemistry. Pickleweed suppressed cordgrass stem density but had no effect on aboveground biomass. For every cordgrass stem lost per square meter, porewater ammonium increased 0.3–1.0 µM. Thus, aboveground competition with pickleweed weakened the effects of cordgrass on sediment biogeochemistry. Predictions about plant–soil feedbacks, especially under future climate scenarios, will be improved when plant–plant interactions are considered, particularly those containing dominant and foundation species.