Unlocking the power of AI models: exploring protein folding prediction through comparative analysis.

Protein structure determination has made progress with the aid of deep learning models, enabling the prediction of protein folding from protein sequences. However, obtaining accurate predictions becomes essential in certain cases where the protein structure remains undescribed. This is particularly challenging when dealing with rare, diverse structures and complex sample preparation. Different metrics assess prediction reliability and offer insights into result strength, providing a comprehensive understanding of protein structure by combining different models. In a previous study, two proteins named ARM58 and ARM56 were investigated. These proteins contain four domains of unknown function and are present in Leishmania spp. ARM refers to an antimony resistance marker. The study's main objective is to assess the accuracy of the model's predictions, thereby providing insights into the complexities and supporting metrics underlying these findings. The analysis also extends to the comparison of predictions obtained from other species and organisms. Notably, one of these proteins shares an ortholog with Trypanosoma cruzi and Trypanosoma brucei, leading further significance to our analysis. This attempt underscored the importance of evaluating the diverse outputs from deep learning models, facilitating comparisons across different organisms and proteins. This becomes particularly pertinent in cases where no previous structural information is available.

Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.

Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba-/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb-/- and Ncf1-/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba-/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

Cyba-deficient mice display an increase in hematopoietic stem cells and an overproduction of immunoglobulins.

The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.

PCNA Deubiquitylases Control DNA Damage Bypass at Replication Forks.

DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNA-linked ubiquitin chains. Ubp10 and Ubp12 associate with replication forks in a fashion determined by Ubp10 dependency on lagging-strand PCNA residence, and they downregulate translesion polymerase recruitment and template switch events engaging nascent strands. These findings reveal PCNAK164 deubiquitylation as a key mechanism for the modulation of lesion bypass during replication, which might set a framework for establishing strand-differential pathway choices. We propose that damage tolerance is tempered at replication forks to limit the extension of bypass events and sustain chromosome replication rates.

Analysis of Cohesin Association to Newly Replicated DNA Through Nascent Strand Binding Assay (NSBA).

Replication forks engage chromatin-bound cohesin complexes during chromosome replication. Interfacing between cohesin and replication forks influences both cohesion establishment and fork functionality. However, the mechanisms mediating this process are scarcely understood. Here we describe the nascent strand binding assay (NSBA) methodology, developed in budding yeast to discriminate the association of cohesin to either parental unreplicated or nascent DNA in the environment of replication forks. NSBA quantitatively estimates the association of a protein of interest to newly replicated DNA. For this, nascent strands are in vivo labeled with the thymine analogue bromodeoxyuridine and chromatin is immunoprecipitated to isolate a fraction enriched in DNA associated to the target protein. The abundance of nascent DNA is then assessed through BrdU immunoprecipitation followed by quantitative PCR, allowing for the parallel analysis of diverse genomic regions. While originally employed to characterize the association of cohesin to nascent sister chromatids, NSBA can be applied to study other factors dynamically associating to nascent DNA.

SHP1 and SHP2 inhibition enhances the pro-differentiative effect of phorbol esters: an alternative approach against acute myeloid leukemia.

BACKGROUND: The differentiation-based therapy for acute promyelocytic leukemia (APL) is an inspiring example for the search of novel strategies aimed at treatment of other subtypes of acute myeloid leukemia (AML). Thus, the discovery of new molecular players in cell differentiation becomes a paramount research area to achieve this goal. Here, the involvement of the protein tyrosine phosphatases SHP1 and SHP2 on leukemic cells differentiation is shown, along with the therapeutic possibilities of their targeting to enhance the differentiation induction effect of phorbol esters. METHODS: The oxidation status and enzymatic activity of SHP1 and SHP2 during PMA-induced differentiation of HEL cells was evaluated. Additionally, the effects of RNAi-mediated downregulation of these phosphatases on cell differentiation was studied. Afterwards, the impact of chemical inhibition of SHP1 and SHP2 on differentiation both in the presence and absence of phorbol esters was tested. Finally, the anti-leukemic potential of phorbol esters and chemical inhibitors of SHP1 and SHP2 was addressed in several AML model cell lines, a xenograft mouse model and AML primary cells in vitro. RESULTS: An increase of oxidation with a concomitant decrease of activity was observed for both phosphatases at the onset of PMA-induced differentiation. Consistently, silencing of these proteins favored the process, with an enhanced effect upon their simultaneous downregulation. Moreover, the proteins SRC and ß-catenin were identified as downstream targets of SHP1 and SHP2 in this context. In agreement with these findings, chemical inhibition of the phosphatases promoted cell differentiation itself and enhanced the effect of phorbol esters. Interestingly, treatment with the phorbol ester prostratin and the dual inhibitor of SHP1 and SHP2 NSC87877 synergistically hampered the proliferation of AML cell lines, prolonged the survival of xenografted mice and reduced the clonogenic potential of AML primary cells. CONCLUSIONS: SHP1 and SHP2 are relevant mediators of differentiation in AML cells and their inhibition either alone or in combination with prostratin seems a promising differentiation-based therapeutic strategy against different subtypes of AML beyond APL.

Replisome-Cohesin Interfacing: A Molecular Perspective.

Cohesion is established in S-phase through the action of key replisome factors as replication forks engage cohesin molecules. By holding sister chromatids together, cohesion critically assists both an equal segregation of the duplicated genetic material and an efficient repair of DNA breaks. Nonetheless, the molecular events leading the entrapment of nascent chromatids by cohesin during replication are only beginning to be understood. The authors describe here the essential structural features of the cohesin complex in connection to its ability to associate DNA molecules and review the current knowledge on the architectural-functional organization of the eukaryotic replisome, significantly advanced by recent biochemical and structural studies. In light of this novel insight, the authors discuss the mechanisms proposed to assist interfacing of replisomes with chromatin-bound cohesin complexes and elaborate on models for nascent chromatids entrapment by cohesin in the environment of the replication fork.

Reactive oxygen species in haematopoiesis: leukaemic cells take a walk on the wild side.

Oxidative stress is related to ageing and degenerative diseases, including cancer. However, a moderate amount of reactive oxygen species (ROS) is required for the regulation of cellular signalling and gene expression. A low level of ROS is important for maintaining quiescence and the differentiation potential of haematopoietic stem cells (HSCs), whereas the level of ROS increases during haematopoietic differentiation; thus, suggesting the importance of redox signalling in haematopoiesis. Here, we will analyse the importance of ROS for haematopoiesis and include evidence showing that cells from leukaemia patients live under oxidative stress. The potential sources of ROS will be described. Finally, the level of oxidative stress in leukaemic cells can also be harnessed for therapeutic purposes. In this regard, the reliance of front-line anti-leukaemia chemotherapeutics on increased levels of ROS for their mechanism of action, as well as the active search for novel compounds that modulate the redox state of leukaemic cells, will be analysed.

Cohesin dynamic association to chromatin and interfacing with replication forks in genome integrity maintenance.

Proliferating cells need to accurately duplicate and pass their genetic material on to daughter cells. Problems during replication and partition challenge the structural and numerical integrity of chromosomes. Diverse mechanisms, as the DNA replication checkpoint, survey the correct progression of replication and couple it with other cell cycle events to preserve genome integrity. The structural maintenance of chromosomes (SMC) cohesin complex primarily contributes to chromosome duplication by mediating the tethering of newly replicated sister chromatids, thus assisting their equal segregation in mitosis. In addition, cohesin exerts important functions in genome organization, gene expression and DNA repair. These are determined by cohesin's ability to bring together different DNA segments and, hence, by the fashion and dynamics of its interaction with chromatin. It recently emerged that cohesin contributes to the protection of stalled replication forks through a mechanism requiring its timely mobilization from unreplicated DNA and relocation to nascent strands. This mechanism relies on DNA replication checkpoint-dependent cohesin ubiquitylation and promotes nascent sister chromatid entrapment, likely contributing to preserve stalled replisome-fork architectural integrity. Here we review how cohesin dynamic association to chromatin is controlled through post-translational modifications to dictate its functions during chromosome duplication. We also discuss recent insights on the mechanism that mediates interfacing of replisome components with chromatin-bound cohesin and its contribution to the establishment of sister chromatid cohesion and the protection of stalled replication forks.

Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics.

Replication fork integrity is challenged in conditions of stress and protected by the Mec1/ATR checkpoint to preserve genome stability. Still poorly understood in fork protection is the role played by the structural maintenance of chromosomes (SMC) cohesin complex. We uncovered a role for the Rsp5Bul2 ubiquitin ligase in promoting survival to replication stress by preserving stalled fork integrity. Rsp5Bul2 physically interacts with cohesin and the Mec1 kinase, thus promoting checkpoint-dependent cohesin ubiquitylation and cohesin-mediated fork protection. Ubiquitylation mediated by Rsp5Bul2 promotes cohesin mobilization from chromatin neighboring stalled forks, likely by stimulating the Cdc48/p97 ubiquitin-selective segregase, and its timely association to nascent chromatids. This Rsp5Bul2 fork protection mechanism requires the Wpl1 cohesin mobilizer as well as the function of the Eco1 acetyltransferase securing sister chromatid entrapment. Our data indicate that ubiquitylation facilitates cohesin dynamic interfacing with replication forks within a mechanism preserving stalled-fork functional architecture.

PP2A Controls Genome Integrity by Integrating Nutrient-Sensing and Metabolic Pathways with the DNA Damage Response.

Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.

The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis.

Eukaryotic cells have to cope with the constant generation of reactive oxygen species (ROS). Although the excessive production of ROS might be deleterious for cell biology, there is a plethora of evidence showing that moderate levels of ROS are important for the control of cell signaling and gene expression. The family of the nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases or Nox) has evolved to produce ROS in response to different signals; therefore, they fulfil a central role in the control of redox signaling. The role of NADPH oxidases in vascular physiology has been a field of intense study over the last two decades. In this review we will briefly analyze how ROS can regulate signaling and gene expression. We will address the implication of NADPH oxidases and redox signaling in angiogenesis, and finally, the therapeutic possibilities derived from this knowledge will be discussed.

The Multiple Roles of Ubiquitylation in Regulating Challenged DNA Replication.

DNA replication is essential for the propagation of life and the development of complex organisms. However, replication is a risky process as it can lead to mutations and chromosomal alterations. Conditions challenging DNA synthesis by replicative polymerases or DNA helix unwinding, generally termed as replication stress, can halt replication fork progression. Stalled replication forks are unstable, and mechanisms exist to protect their integrity, which promote an efficient restart of DNA synthesis and counteract fork collapse characterized by the accumulation of DNA lesions and mutagenic events. DNA replication is a highly regulated process, and several mechanisms control replication timing and integrity both during unperturbed cell cycles and in response to replication stress. Work over the last two decades has revealed that key steps of DNA replication are controlled by conjugation of the small peptide ubiquitin. While ubiquitylation was traditionally linked to protein degradation, the complexity and flexibility of the ubiquitin system in regulating protein function have recently emerged. Here we review the multiple roles exerted by ubiquitin-conjugating enzymes and ubiquitin-specific proteases, as well as readers of ubiquitin chains, in the control of eukaryotic DNA replication and replication-coupled DNA damage tolerance and repair.

Nucleolytic processing of aberrant replication intermediates by an Exo1-Dna2-Sae2 axis counteracts fork collapse-driven chromosome instability.

Problems during DNA replication underlie genomic instability and drive malignant transformation. The DNA damage checkpoint stabilizes stalled replication forks thus counteracting aberrant fork transitions, DNA breaks and chromosomal rearrangements. We analyzed fork processing in checkpoint deficient cells by coupling psoralen crosslinking with replication intermediate two-dimensional gel analysis. This revealed a novel role for Exo1 nuclease in resecting reversed replication fork structures and counteracting the accumulation of aberrant intermediates resembling fork cleavage products. Genetic analyses demonstrated a functional interplay of Exo1 with Mus81, Dna2 and Sae2 nucleases in promoting cell survival following replication stress, suggestive of concerted nucleolytic processing of stalled forks. While Mus81 and other Structure Specific Endonucleases do not contribute to obvious collapsed fork transitions, Dna2 promotes reversed fork resection likely by facilitating Exo1 access to nascent strands. Instead, Sae2 cooperates with Exo1 in counteracting putative fork cleavage events linked to double strand breaks formation and increased gross chromosomal rearrangement rates. Our data indicate that in checkpoint deficient cells diverse nuclease activities interface to eliminate aberrant replication intermediates and prevent chromosome instability.

PTPN13 and ß-Catenin Regulate the Quiescence of Hematopoietic Stem Cells and Their Interaction with the Bone Marrow Niche.

The regulation of hematopoietic stem cells (HSCs) depends on the integration of the multiple signals received from the bone marrow niche. We show the relevance of the protein tyrosine phosphatase PTPN13 and ß-catenin as intracellular signaling molecules to control HSCs adhesiveness, cell cycling, and quiescence. Lethally irradiated mice transplanted with Lin(-) bone marrow cells in which PTPN13 or ß-catenin had been silenced showed a significant increase of long-term (LT) and short-term (ST) HSCs. A decrease in cycling cells was also found, together with an increase in quiescence. The decreased expression of PTPN13 or ß-catenin was linked to the upregulation of several genes coding for integrins and several cadherins, explaining the higher cell adhesiveness. Our data are consistent with the notion that the levels of PTPN13 and ß-catenin must be strictly regulated by extracellular signaling to regulate HSC attachment to the niche and the balance between proliferation and quiescence.

PTPN13 regulates cellular signalling and ß-catenin function during megakaryocytic differentiation.

PTPN13 is a high-molecular weight intracellular phosphatase with several isoforms that exhibits a highly modular structure. Although in recent years different roles have been described for PTPN13, we are still far from understanding its function in cell biology. Here we show that PTPN13 expression is activated during megakaryocytic differentiation at the protein and mRNA level. Our results show that the upregulation of PTPN13 inhibits megakaryocytic differentiation, while PTPN13 silencing triggers differentiation. The ability of PTPN13 to alter megakaryocytic differentiation can be explained by its capacity to regulate ERK and STAT signalling. Interestingly, the silencing of ß-catenin produced the same effect as PTPN13 downregulation. We demonstrate that both proteins coimmunoprecipitate and colocalise. Moreover, we provide evidence showing that PTPN13 can regulate ß-catenin phosphorylation, stability and transcriptional activity. Therefore, the ability of PTPN13 to control megakaryocytic differentiation must be intimately linked to the regulation of ß-catenin function. Moreover, our results show for the first time that PTPN13 is stabilised upon Wnt signalling, which makes PTPN13 an important player in canonical Wnt signalling. Our results show that PTPN13 behaves as an important regulator of megakaryocytic differentiation in cell lines and also in murine haematopoietic progenitors. This importance can be explained by the ability of PTPN13 to regulate cellular signalling, and especially through the regulation of ß-catenin stability and function. Our results hold true for different megakaryocytic cell lines and also for haematopoietic progenitors, suggesting that these two proteins may play a relevant role during in vivo megakaryopoiesis.

NADPH oxidases as therapeutic targets in chronic myelogenous leukemia.

PURPOSE: Cancer cells show higher levels of reactive oxygen species (ROS) than normal cells and increasing intracellular ROS levels are becoming a recognized strategy against tumor cells. Thus, diminishing ROS levels could be also detrimental to cancer cells. We surmise that avoiding ROS generation would be a better option than quenching ROS with antioxidants. Chronic myelogenous leukemia (CML) is triggered by the expression of BCR-ABL kinase, whose activity leads to increased ROS production, partly through NADPH oxidases. Here, we assessed NADPH oxidases as therapeutic targets in CML. EXPERIMENTAL DESIGN: We have analyzed the effect of different NADPH oxidase inhibitors, either alone or in combination with BCR-ABL inhibitors, in CML cells and in two different animal models for CML. RESULTS: NADPH oxidase inhibition dramatically impaired the proliferation and viability of BCR-ABL-expressing cells due to the attenuation of BCR-ABL signaling and a pronounced cell-cycle arrest. Moreover, the combination of NADPH oxidase inhibitors with BCR-ABL inhibitors was highly synergistic. Two different animal models underscore the effectiveness of NADPH oxidase inhibitors and their combination with BCR-ABL inhibitors for CML targeting in vivo. CONCLUSION: Our results offer further therapeutic opportunities for CML, by targeting NADPH oxidases. In the future, it would be worthwhile conducting further experiments to ascertain the feasibility of translating such therapies to clinical practice.

DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

The DNA damage checkpoint response to replication stress: A Game of Forks.

Conditions challenging replication fork progression, collectively referred to as replication stress, represent a major source of genomic instability and are associated to cancer onset. The replication checkpoint, a specialized branch of the DNA damage checkpoint, monitors fork problems, and triggers a cellular response aimed at preserving genome integrity. Here, we review the mechanisms by which the replication checkpoint monitors and responds to replication stress, focusing on the checkpoint-mediated pathways contributing to protect replication fork integrity. We discuss how cells achieve checkpoint signaling inactivation once replication stress is overcome and how a failure to timely revert checkpoint-mediated changes in cellular physiology might impact on replication dynamics and genome integrity. We also highlight the checkpoint function as an anti-cancer barrier preventing cells malignant transformation following oncogene-induced replication stress.