Controlled modulation of the dynamics of the Deinococcus grandis Dps N‐terminal tails by divalent metals

DNA‐binding proteins from starved cells (Dps) are small multifunctional nanocages expressed by prokaryotes in acute oxidative stress conditions or during the starvation‐induced stationary phase, as a bacterial defense mechanism. Dps proteins protect bacterial DNA from damage by either direct binding or by removing precursors of reactive oxygen species from solution. The DNA‐binding properties of most Dps proteins studied so far are related to their unordered, flexible, N‐ and C‐terminal extensions. In a previous work, we revealed that the N‐terminal tails of Deinoccocus grandis Dps shift from an extended to a compact conformation depending on the ionic strength of the buffer and detected a novel high‐spin ferrous iron center in the proximal ends of those tails. In this work, we further explore the conformational dynamics of the protein by probing the effect of divalent metals binding to the tail by comparing the metal‐binding properties of the wild‐type protein with a binding site‐impaired D34A variant using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism, and small‐angle X‐ray scattering. The N‐terminal ferrous species was also characterized by Mössbauer spectroscopy. The results herein presented reveal that the conformation of the N‐terminal tails is altered upon metal binding in a gradual, reversible, and specific manner. These observations may point towards the existence of a regulatory process for the DNA‐binding properties of Dps proteins through metal binding to their N‐ and/or C‐terminal extensions.     

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal‐to‐zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc‐miR‐205 and bovine bta‐miR‐10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc‐miR‐205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte‐to‐embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.     

The PAX5‐JAK2 translocation acts as dual‐hit mutation that promotes aggressive B‐cell leukemia via nuclear STAT5 activation

While PAX5 is an important tumor suppressor gene in B‐cell acute lymphoblastic leukemia (B‐ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5‐JAK2 encodes a protein consisting of the PAX5 DNA‐binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5‐JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5 ^Jak2/+ mice rapidly developed an aggressive B‐ALL in the absence of another cooperating exogenous gene mutation. The DNA‐binding function and kinase activity of Pax5‐Jak2 as well as IL‐7 signaling contributed to leukemia development. Interestingly, all Pax5 ^Jak2/+ tumors lost the remaining wild‐type Pax5 allele, allowing efficient DNA‐binding of Pax5‐Jak2. While we could not find evidence for a nuclear role of Pax5‐Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5 ^Jak2/+ B‐ALL tumors, implying that nuclear Pax5‐Jak2 phosphorylates STAT5. Together, these data reveal Pax5‐Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.     

BRCA2 promotes DNA‐RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair

The BRCA2 tumor suppressor is a DNA double‐strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2‐deficient cells spontaneously accumulate DNA‐RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD‐box RNA helicase DDX5 as a BRCA2‐interacting protein. DDX5 associates with DNA‐RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA‐RNA hybrid‐unwinding activity of DDX5 helicase. An impaired BRCA2‐DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2‐T207A, reduces the association of DDX5 with DNA‐RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA‐RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.     

Structural investigation of CDCA3‐Cdh1 protein–protein interactions using in vitro studies and molecular dynamics simulation

The anaphase‐promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell‐cycle proteins and their functions during mitosis. Levels of the protein cell division cycle‐associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/C^Cdh1. CDCA3 is an intrinsically disordered protein and contains both C‐terminal KEN box and D‐box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation‐dependent non‐canonical ABBA‐like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA‐like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H‐bonds, hydrophobic and ionic interactions across the CDCA3 ABBA‐like motif in the linker region between KEN and D‐box motifs. This linker region adopts a well‐defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non‐canonical ABBA‐like motif to promote binding to the APC/C^Cdh1 and regulation of CDCA3 protein levels.     

ScrepYard: An online resource for disulfide‐stabilized tandem repeat peptides

Receptor avidity through multivalency is a highly sought‐after property of ligands. While readily available in nature in the form of bivalent antibodies, this property remains challenging to engineer in synthetic molecules. The discovery of several bivalent venom peptides containing two homologous and independently folded domains (in a tandem repeat arrangement) has provided a unique opportunity to better understand the underpinning design of multivalency in multimeric biomolecules, as well as how naturally occurring multivalent ligands can be identified. In previous work, we classified these molecules as a larger class termed secreted cysteine‐rich repeat‐proteins (SCREPs). Here, we present an online resource; ScrepYard, designed to assist researchers in identification of SCREP sequences of interest and to aid in characterizing this emerging class of biomolecules. Analysis of sequences within the ScrepYard reveals that two‐domain tandem repeats constitute the most abundant SCREP domain architecture, while the interdomain “linker” regions connecting the functional domains are found to be abundant in amino acids with short or polar sidechains and contain an unusually high abundance of proline residues. Finally, we demonstrate the utility of ScrepYard as a virtual screening tool for discovery of putatively multivalent peptides, by using it as a resource to identify a previously uncharacterized serine protease inhibitor and confirm its predicted activity using an enzyme assay.     

EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

Nasopharyngeal carcinoma (NPC) is an Epstein‐Barr virus (EBV)‐associated epithelial malignancy. The high expression of BART‐miRNAs (miR‐BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART‐miRNAs work co‐operatively to modulate the DNA damage response (DDR) by reducing Ataxia‐telangiectasia‐mutated (ATM) activity. In this study, we further investigated the role of miR‐BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down‐regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2‐3p, BART12, BART17‐5p and BART19‐3p in BRCA1 expression. The ectopic expression of these four miR‐BARTs suppressed endogenous BRCA1 expression in EBV‐negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR‐BARTs activities in C666‐1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR‐BART17‐5p and miR‐BART19‐3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA‐damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR‐BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.     

The free energy folding penalty accompanying binding of intrinsically disordered α‐helical motifs

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes and preform critical roles in many cellular processes, most often through the association with globular proteins. Despite lacking a stable three‐dimensional structure by themselves, they may acquire a defined conformation upon binding globular targets. The most common type of secondary structure acquired by these binding motifs entails formation of an α‐helix. It has been hypothesized that such disorder‐to‐order transitions are associated with a significant free energy penalty due to IDP folding, which reduces the overall IDP‐target affinity. However, the exact magnitude of IDP folding penalty in α‐helical binding motifs has not been systematically estimated. Here, we report the folding penalty contributions for 30 IDPs undergoing folding‐upon‐binding and find that the average IDP folding penalty is +2.0 kcal/mol and ranges from 0.7 to 3.5 kcal/mol. We observe that the folding penalty scales approximately linearly with the change in IDP helicity upon binding, which provides a simple empirical way to estimate folding penalty. We analyze to what extent do pre‐structuring and target‐bound IDP dynamics (fuzziness) reduce the folding penalty and find that these effects combined, on average, reduce the folding cost by around half. Taken together, the presented analysis provides a quantitative basis for understanding the role of folding penalty in IDP‐target interactions and introduces a method estimate this quantity. Estimation and reduction of IDP folding penalty may prove useful in the rational design of helix‐stabilized inhibitors of IDP‐target interactions.StatementThe α‐helical binding motifs are ubiquitous among the intrinsically disordered proteins (IDPs). Upon binding their targets, they undergo a disorder‐to‐order transition, which is accompanied by a significant folding penalty whose magnitude is generally not known. Here, we use recently developed statistical‐thermodynamic model to estimate the folding penalties for 30 IDPs and clarify the roles of IDP pre‐folding and bound‐state dynamics in reducing the folding penalty.     

Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF_Y. CNF_Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF_Y, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.     

A new linear cyclin docking motif that mediates exclusively S‐phase CDK‐specific signaling

Cyclin‐dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation‐dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S‐phase CDK (S‐CDK) Clb5/6‐Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine‐tuning of cell cycle events. Using time‐lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation‐driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL‐containing but not of NLxxxL‐containing substrates. The NLxxxL motif was found to govern S‐cyclin‐specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.     

A retrospective analysis of EBV‐DNA status with the prognosis of lymphoma

Epstein–Barr virus (EBV) infection is proved to be associated with clinicopathology of lymphoma. However, little is known about the relationship between EBV‐DNA status after treatment and prognosis. In this study, real‐time polymerase chain reaction (PCR) was used for quantitative detection of EBV‐DNA load in peripheral blood of all 26,527 patients with lymphoma, and the clinical characteristics and prognosis of 202 patients were retrospectively analysed, including 100 patients with positive EBV‐DNA and 102 randomly selected patients with negative EBV‐DNA. We found that the average rate of EBV‐DNA positivity in lymphomas was 0.376%, and EBV‐DNA‐positive patients presented higher risk with elevated lactate dehydrogenase (LDH) and β2‐MG level, B symptoms, secondary hemophagocytic syndrome and lower objective response rate compared to EBV‐DNA‐negative patients. Multivariate analysis revealed EBV‐DNA‐positive patients had inferior progression‐free survival (PFS) and overall survival (OS) and EBV‐DNA level before treatment was related to PFS but not OS of T/NK cell lymphoma. In T/NK cell lymphoma, EBV‐DNA converting negative after treatment was correlated with better PFS but not OS, and second‐line therapy could induce more EBV‐DNA‐negative conversion compared to CHOP‐based therapy. In all, EBV‐DNA positivity before treatment can be a biomarker representing the tumour burden and an independent prognostic factor. EBV‐DNA‐negative conversion after treatment is a good prognostic factor for T/NK cell lymphomas.     

Comparative proteome analysis identified CD44 as a possible serum marker for docetaxel resistance in castration‐resistant prostate cancer

Baseline or acquired resistance to docetaxel (DOC) represents a significant risk for patients with metastatic prostate cancer (PC). In the last years, novel therapy regimens have been approved providing reasonable alternatives for DOC‐resistant patients making prediction of DOC resistance of great clinical importance. We aimed to identify serum biomarkers, which are able to select patients who will not benefit from DOC treatment. DOC‐resistant PC3‐DR and DU145‐DR sublines and their sensitive parental cell lines (DU145, PC3) were comparatively analyzed using liquid chromatography‐coupled tandem mass spectrometry (LC‐MS/MS). Results were filtered using bioinformatics approaches to identify promising serum biomarkers. Serum levels of five proteins were determined in serum samples of 66 DOC‐treated metastatic castration‐resistant PC patients (mCRPC) using ELISA. Results were correlated with clinicopathological and survival data. CD44 was subjected to further functional cell culture analyses. We found at least 177 two‐fold significantly overexpressed proteins in DOC‐resistant cell lines. Our bioinformatics method suggested 11/177 proteins to be secreted into the serum. We determined serum levels of five (CD44, MET, GSN, IL13RA2 and LNPEP) proteins in serum samples of DOC‐treated patients and found high CD44 serum levels to be independently associated with poor overall survival (p = 0.001). In accordance, silencing of CD44 in DU145‐DR cells resulted in re‐sensitization to DOC. In conclusion, high serum CD44 levels may help identify DOC‐resistant patients and may thereby help optimize clinical decision‐making regarding type and timing of therapy for mCRPC patients. In addition, our in vitro results imply the possible functional involvement of CD44 in DOC resistance.     

Neural network‐derived Potts models for structure‐based protein design using backbone atomic coordinates and tertiary motifs

Designing novel proteins to perform desired functions, such as binding or catalysis, is a major goal in synthetic biology. A variety of computational approaches can aid in this task. An energy‐based framework rooted in the sequence‐structure statistics of tertiary motifs (TERMs) can be used for sequence design on predefined backbones. Neural network models that use backbone coordinate‐derived features provide another way to design new proteins. In this work, we combine the two methods to make neural structure‐based models more suitable for protein design. Specifically, we supplement backbone‐coordinate features with TERM‐derived data, as inputs, and we generate energy functions as outputs. We present two architectures that generate Potts models over the sequence space: TERMinator, which uses both TERM‐based and coordinate‐based information, and COORDinator, which uses only coordinate‐based information. Using these two models, we demonstrate that TERMs can be utilized to improve native sequence recovery performance of neural models. Furthermore, we demonstrate that sequences designed by TERMinator are predicted to fold to their target structures by AlphaFold. Finally, we show that both TERMinator and COORDinator learn notions of energetics, and these methods can be fine‐tuned on experimental data to improve predictions. Our results suggest that using TERM‐based and coordinate‐based features together may be beneficial for protein design and that structure‐based neural models that produce Potts energy tables have utility for flexible applications in protein science.     

Structural mechanism for modulation of functional amyloid and biofilm formation by Staphylococcal Bap protein switch

The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca²⁺]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation‐prone region of Staphylococcus aureus Bap which adopts a dumbbell‐shaped fold. The middle module (MM) connecting the N‐terminal and C‐terminal lobes consists of a tandem of novel double‐Ca²⁺‐binding motifs involved in cooperative interaction networks, which undergoes Ca²⁺‐dependent order–disorder conformational switches. The N‐terminal lobe is sufficient to mediate amyloid aggregation through liquid–liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca²⁺] but inhibited by ordered MM stabilized by Ca²⁺ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti‐biofilm drug design.