Extension of a de novo TIM barrel with a rationally designed secondary structure element

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four‐fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub‐problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high‐resolution X‐ray structure for one variant and compare it to our design model. The successful extension of this robust TIM‐barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.     

The roles of highly conserved,non‐catalytic residues in class A β‐lactamases

Evolution minimizes the number of highly conserved amino acid residues in proteins to ensure evolutionary robustness and adaptability. The roles of all highly conserved, non‐catalytic residues, 11% of all residues, in class A β‐lactamase were analyzed by studying the effect of 146 mutations on in cell and in vitro activity, folding, structure, and stability. Residues around the catalytic residues (second shell) contribute to fine‐tuning of the active site structure. Mutations affect the structure over the entire active site and can result in stable but inactive protein. Conserved residues farther away (third shell) ensure a favorable balance of folding versus aggregation or stabilize the folded form over the unfolded state. Once folded, the mutant enzymes are stable and active and show only localized structural effects. These residues are found in clusters, stapling secondary structure elements. The results give an integral picture of the different roles of essential residues in enzymes.     

Protein yoga: Conformational versatility of the Hemolysin II C‐terminal domain detailed by NMR structures for multiple states

The C‐terminal domain of Bacillus cereus hemolysin II (HlyIIC), stabilizes the trans‐membrane‐pore formed by the HlyII toxin and may aid in target cell recognition. Initial efforts to determine the NMR structure of HlyIIC were hampered by cis/trans isomerization about the single proline at position 405 that leads to doubling of NMR resonances. We used the mutant P405M‐HlyIIC that eliminates the cis proline to determine the NMR structure of the domain, which revealed a novel fold. Here, we extend earlier studies to the NMR structure determination of the cis and trans states of WT‐HlyIIC that exist simultaneously in solution. The primary structural differences between the cis and trans states are in the loop that contains P405, and structurally adjacent loops. Thermodynamic linkage analysis shows that at 25 C the cis proline, which already has a large fraction of 20% in the unfolded protein, increases to 50% in the folded state due to coupling with the global stability of the domain. The P405M or P405A substitutions eliminate heterogeneity due to proline isomerization but lead to the formation of a new dimeric species. The NMR structure of the dimer shows that it is formed through domain‐swapping of strand β5, the last segment of secondary structure following P405. The presence of P405 in WT‐HlyIIC strongly disfavors the dimer compared to the P405M‐HlyIIC or P405A‐HlyIIC mutants. The WT proline may thus act as a “gatekeeper,” warding off aggregative misfolding.     

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.     

Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family

Ribonucleotide reductases (RNRs) are used by all free‐living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical‐based mechanism for nucleotide reduction. In this work, we expand on our recent phylogenetic inference of the entire RNR family and describe the evolutionarily relatedness of insertions and extensions around the structurally homologous catalytic barrel. Using evo‐velocity and sequence similarity network (SSN) analyses, we show that the N‐terminal regulatory motif known as the ATP‐cone domain was likely inherited from an ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, we also show that the C‐terminal extensions of class II RNRs can contain folded domains that share homology with an Fe‐S cluster assembly protein. Finally, using sequence analysis and AlphaFold2, we show that the sequence motif of a catalytically essential insertion known as the finger loop is tightly coupled to the catalytic mechanism. Based on these results, we propose an evolutionary model for the diversification of the RNR family.     

A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the α7β1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-α7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

Temporal and brain region‐specific elevations of soluble Amyloid‐β40‐42 in the Ts65Dn mouse model of Down syndrome and Alzheimer’s disease

Down syndrome (DS) is a leading cause of intellectual disability that also results in hallmark Alzheimer''s disease (AD) pathologies such as amyloid beta (Aβ) plaques and hyperphosphorylated tau. The Ts65Dn mouse model is commonly used to study DS, as trisomic Ts65Dn mice carry 2/3 of the triplicated gene homologues as occur in human DS. The Ts65Dn strain also allows investigation of mechanisms common to DS and AD pathology, with many of these triplicated genes implicated in AD; for example, trisomic Ts65Dn mice overproduce amyloid precursor protein (APP), which is then processed into soluble Aβ_40‐42 fragments. Notably, Ts65Dn mice show alterations to the basal forebrain, which parallels the loss of function in this region observed in DS and AD patients early on in disease progression. However, a complete picture of soluble Aβ_40‐42 accumulation in a region‐, age‐, and sex‐specific manner has not yet been characterized in the Ts65Dn model. Here, we show that trisomic mice accumulate soluble Aβ_40‐42 in the basal forebrain, frontal cortex, hippocampus, and cerebellum in an age‐specific manner, with elevation in the frontal cortex and hippocampus as early as 4 months of age. Furthermore, we detected sex differences in accumulation of Aβ_40‐42 within the basal forebrain, with females having significantly higher Aβ_40‐42 at 7–8 months of age. Lastly, we show that APP expression in the basal forebrain and hippocampus inversely correlates with Aβ_40‐42 levels. This spatial and temporal characterization of soluble Aβ_40‐42 in the Ts65Dn model allows for further exploration of the role soluble Aβ plays in the progression of other AD‐like pathologies in these key brain regions.     

IGFBP1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

There is great interest in therapeutically harnessing endogenous regenerative mechanisms to increase the number of β cells in people with diabetes. By performing whole‐genome expression profiling of zebrafish islets, we identified 11 secreted proteins that are upregulated during β‐cell regeneration. We then tested the proteins'' ability to potentiate β‐cell regeneration in zebrafish at supraphysiological levels. One protein, insulin‐like growth factor (Igf) binding‐protein 1 (Igfbp1), potently promoted β‐cell regeneration by potentiating α‐ to β‐cell transdifferentiation. Using various inhibitors and activators of the Igf pathway, we show that Igfbp1 exerts its regenerative effect, at least partly, by inhibiting Igf signaling. Igfbp1''s effect on transdifferentiation appears conserved across species: Treating mouse and human islets with recombinant IGFBP1 in vitro increased the number of cells co‐expressing insulin and glucagon threefold. Moreover, a prospective human study showed that having high IGFBP1 levels reduces the risk of developing type‐2 diabetes by more than 85%. Thus, we identify IGFBP1 as an endogenous promoter of β‐cell regeneration and highlight its clinical importance in diabetes.     

Genetic signature of human longevity in PKC and NF‐κB signaling

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer''s disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans.     

Modulation of phagosome phosphoinositide dynamics by a Legionella phosphoinositide 3‐kinase

The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4‐phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella‐containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3‐kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3‐phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P‐binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.     

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (β/⍺)₈-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.     

MIG‐6 is essential for promoting glucose metabolic reprogramming and tumor growth in triple‐negative breast cancer

Treatment of triple‐negative breast cancer (TNBC) remains challenging due to a lack of effective targeted therapies. Dysregulated glucose uptake and metabolism are essential for TNBC growth. Identifying the molecular drivers and mechanisms underlying the metabolic vulnerability of TNBC is key to exploiting dysregulated cancer metabolism for therapeutic applications. Mitogen‐inducible gene‐6 (MIG‐6) has long been thought of as a feedback inhibitor that targets activated EGFR and suppresses the growth of tumors driven by constitutive activated mutant EGFR. Here, our bioinformatics and histological analyses uncover that MIG‐6 is upregulated in TNBC and that MIG‐6 upregulation is positively correlated with poorer clinical outcomes in TNBC. Metabolic arrays and functional assays reveal that MIG‐6 drives glucose metabolism reprogramming toward glycolysis. Mechanistically, MIG‐6 recruits HAUSP deubiquitinase for stabilizing HIF1α protein expression and the subsequent upregulation of GLUT1 and other HIF1α‐regulated glycolytic genes, substantiating the comprehensive regulation of MIG‐6 in glucose metabolism. Moreover, our mouse studies demonstrate that MIG‐6 regulates GLUT1 expression in tumors and subsequent tumor growth in vivo. Collectively, this work reveals that MIG‐6 is a novel prognosis biomarker, metabolism regulator, and molecular driver of TNBC.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.     

Histidine dephosphorylation of the Gβ protein GPB‐1 promotes axon regeneration in C. elegans

Histidine phosphorylation is an emerging noncanonical protein phosphorylation in animals, yet its physiological role remains largely unexplored. The protein histidine phosphatase (PHPT1) was recently identified for the first time in mammals. Here, we report that PHIP‐1, an ortholog of PHPT1 in Caenorhabditis elegans, promotes axon regeneration by dephosphorylating GPB‐1 Gβ at His‐266 and inactivating GOA‐1 Goα signaling, a negative regulator of axon regeneration. Overexpression of the histidine kinase NDK‐1 also inhibits axon regeneration via GPB‐1 His‐266 phosphorylation. Thus, His‐phosphorylation plays an antiregenerative role in C. elegans. Furthermore, we identify a conserved UNC‐51/ULK kinase that functions in autophagy as a PHIP‐1‐binding protein. We demonstrate that UNC‐51 phosphorylates PHIP‐1 at Ser‐112 and activates its catalytic activity and that this phosphorylation is required for PHIP‐1‐mediated axon regeneration. This study reveals a molecular link from ULK to protein histidine phosphatase, which facilitates axon regeneration by inhibiting trimeric G protein signaling.     

Tubulin Polymerization-promoting Protein (TPPP/p25α) Promotes Unconventional Secretion of α-Synuclein through Exophagy by Impairing Autophagosome-Lysosome Fusion

Aggregation of α-synuclein can be promoted by the tubulin polymerization-promoting protein/p25α, which we have used here as a tool to study the role of autophagy in the clearance of α-synuclein. In NGF-differentiated PC12 catecholaminergic nerve cells, we show that de novo expressed p25α co-localizes with α-synuclein and causes its aggregation and distribution into autophagosomes. However, p25α also lowered the mobility of autophagosomes and hindered the final maturation of autophagosomes by preventing their fusion with lysosomes for the final degradation of α-synuclein. Instead, p25α caused a 4-fold increase in the basal level of α-synuclein secreted into the medium. Secretion was strictly dependent on autophagy and could be up-regulated (trehalose and Rab1A) or down-regulated (3-methyladenine and ATG5 shRNA) by enhancers or inhibitors of autophagy or by modulating minus-end-directed (HDAC6 shRNA) or plus-end-directed (Rab8) trafficking of autophagosomes along microtubules. Finally, we show in the absence of tubulin polymerization-promoting protein/p25α that α-synuclein release was modulated by dominant mutants of Rab27A, known to regulate exocytosis of late endosomal (and amphisomal) elements, and that both lysosomal fusion block and secretion of α-synuclein could be replicated by knockdown of the p25α target, HDAC6, the predominant cytosolic deacetylase in neurons. Our data indicate that unconventional secretion of α-synuclein can be mediated through exophagy and that factors, which increase the pool of autophagosomes/amphisomes (e.g. lysosomal disturbance) or alter the polarity of vesicular transport of autophagosomes on microtubules, can result in an increased release of α-synuclein monomer and aggregates to the surroundings.     

Characterization of early and late transition states of the folding pathway of a SH2 domain

Albeit SH2 domains are abundant protein–protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C‐terminal SH2 domain of SHP2, conducted through a combination of site‐directed mutagenesis and kinetic (un)folding experiments (Φ‐value analysis). The energetic profile of the folding reaction of the C‐SH2 domain is described by a three‐state mechanism characterized by the presence of two transition states and a high‐energy intermediate. The production of 29 site‐directed variants allowed us to calculate the degree of native‐like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ‐values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N‐terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.     

MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti‐tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL‐2 or IL‐15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA‐181a as a key modulator of human γδ T cell differentiation. We observe that miR‐181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR‐181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR‐181a overexpression restricts their levels of NKG2D and TNF‐α. Upon in silico analysis, we identify two miR‐181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR‐181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next‐generation immunotherapies.     

Adiponectin up‐regulates the decrease of myocardial autophagic flux induced by β1‐adrenergic receptor autoantibody partly dependent on AMPK

Cardiomyocytes autophagy is essential for maintaining cardiac function. Our previous studies have found that β₁‐adrenergic receptor autoantibody (β₁‐AA) induced the decreased myocardial autophagic flux, which resulted in cardiomyocyte death and cardiac dysfunction. And other studies demonstrated that β₁‐AA induced the decrease of AMPK phosphorylation, the key hub of autophagy pathway, while adiponectin up‐regulated autophagic flux mediated by AMPK. However, it is not clear whether adiponectin improves the inhibition of myocardial autophagic flux induced by β₁‐AA by up‐regulating the level of AMPK phosphorylation. In this study, it has been confirmed that β₁‐AA induced the decrease of AMPK phosphorylation level in both vivo and vitro. Moreover, pretreatment of cardiomyocytes with AMPK inhibitor Compound C could further reduce the autophagic flux induced by β₁‐AA. Adiponectin deficiency could aggravate the decrease of myocardial AMPK phosphorylation level, autophagic flux and cardiac function induced by β₁‐AA. Further, exogenous adiponectin could reverse the decline of AMPK phosphorylation level and autophagic flux induced by β₁‐AA and even reduce cardiomyocyte death. While pretreated with the Compound C, the adiponectin treatment did not improve the decreased autophagosome formation, but still improved the decreased autophagosome clearance induced by β₁‐AA in cardiomyocytes. This study is the first time to confirm that β₁‐AA could inhibit myocardial autophagic flux by down‐regulating AMPK phosphorylation level. Adiponectin could improve the inhibition of myocardial autophagic flux induced by β₁‐AA partly dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β₁‐AA‐positive cardiac dysfunction.