NADPH oxidase 2 activity disrupts Calmodulin/CaMKIIα complex via redox modifications of CaMKIIα-contained Cys30 and Cys289: Implications in Parkinson's disease.

Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) signaling in the brain plays a critical role in regulating neuronal Ca2+ homeostasis. Its dysfunctional activity is associated with various neurological and neurodegenerative disorders, including Parkinson's disease (PD). Using computational modeling analysis, we predicted that, two essential cysteine residues contained in CaMKIIα, Cys30 and Cys289, may undergo redox modifications impacting the proper functioning of the CaMKIIα docking site for Ca2+/CaM, thus impeding the formation of the CaMKIIα:Ca2+/CaM complex, essential for a proper modulation of CaMKIIα kinase activity. Our subsequent in vitro investigations confirmed the computational predictions, specifically implicating Cys30 and Cys289 residues in impairing CaMKIIα:Ca2+/CaM interaction. We observed CaMKIIα:Ca2+/CaM complex disruption in dopamine (DA) nigrostriatal neurons of post-mortem Parkinson's disease (PD) patients' specimens, addressing the high relevance of this event in the disease. CaMKIIα:Ca2+/CaM complex disruption was also observed in both in vitro and in vivo rotenone models of PD, where this phenomenon was associated with CaMKIIα kinase hyperactivity. Moreover, we observed that, NADPH oxidase 2 (NOX2), a major enzymatic generator of superoxide anion (O2●-) and hydrogen peroxide (H2O2) in the brain with implications in PD pathogenesis, is responsible for CaMKIIα:Ca2+/CaM complex disruption associated to a stable Ca2+CAM-independent CaMKIIα kinase activity and intracellular Ca2+ accumulation. The present study highlights the importance of oxidative stress, in disturbing the delicate balance of CaMKIIα signaling in calcium dysregulation, offering novel insights into PD pathogenesis.

In situ functional cell phenotyping reveals microdomain networks in colorectal cancer recurrence.

Tumors are dynamic ecosystems comprising localized niches (microdomains), possessing distinct compositions and spatial configurations of cancer and non-cancer cell populations. Microdomain-specific network signaling coevolves with a continuum of cell states and functional plasticity associated with disease progression and therapeutic responses. We present LEAPH, an unsupervised machine learning algorithm for identifying cell phenotypes, which applies recursive steps of probabilistic clustering and spatial regularization to derive functional phenotypes (FPs) along a continuum. Combining LEAPH with pointwise mutual information and network biology analyses enables the discovery of outcome-associated microdomains visualized as distinct spatial configurations of heterogeneous FPs. Utilization of an immunofluorescence-based (51 biomarkers) image dataset of colorectal carcinoma primary tumors (n = 213) revealed microdomain-specific network dysregulation supporting cancer stem cell maintenance and immunosuppression that associated selectively with the recurrence phenotype. LEAPH enables an explainable artificial intelligence platform providing insights into pathophysiological mechanisms and novel drug targets to inform personalized therapeutic strategies.

Spatial domain analysis predicts risk of colorectal cancer recurrence and infers associated tumor microenvironment networks.

An unmet clinical need in solid tumor cancers is the ability to harness the intrinsic spatial information in primary tumors that can be exploited to optimize prognostics, diagnostics and therapeutic strategies for precision medicine. Here, we develop a transformational spatial analytics computational and systems biology platform (SpAn) that predicts clinical outcomes and captures emergent spatial biology that can potentially inform therapeutic strategies. We apply SpAn to primary tumor tissue samples from a cohort of 432 chemo-naïve colorectal cancer (CRC) patients iteratively labeled with a highly multiplexed (hyperplexed) panel of 55 fluorescently tagged antibodies. We show that SpAn predicts the 5-year risk of CRC recurrence with a mean AUROC of 88.5% (SE of 0.1%), significantly better than current state-of-the-art methods. Additionally, SpAn infers the emergent network biology of tumor microenvironment spatial domains revealing a spatially-mediated role of CRC consensus molecular subtype features with the potential to inform precision medicine.

Explainable AI (xAI) for Anatomic Pathology.

Pathologists are adopting whole slide images (WSIs) for diagnosis, thanks to recent FDA approval of WSI systems as class II medical devices. In response to new market forces and recent technology advances outside of pathology, a new field of computational pathology has emerged that applies artificial intelligence (AI) and machine learning algorithms to WSIs. Computational pathology has great potential for augmenting pathologists' accuracy and efficiency, but there are important concerns regarding trust of AI due to the opaque, black-box nature of most AI algorithms. In addition, there is a lack of consensus on how pathologists should incorporate computational pathology systems into their workflow. To address these concerns, building computational pathology systems with explainable AI (xAI) mechanisms is a powerful and transparent alternative to black-box AI models. xAI can reveal underlying causes for its decisions; this is intended to promote safety and reliability of AI for critical tasks such as pathology diagnosis. This article outlines xAI enabled applications in anatomic pathology workflow that improves efficiency and accuracy of the practice. In addition, we describe HistoMapr-Breast, an initial xAI enabled software application for breast core biopsies. HistoMapr-Breast automatically previews breast core WSIs and recognizes the regions of interest to rapidly present the key diagnostic areas in an interactive and explainable manner. We anticipate xAI will ultimately serve pathologists as an interactive computational guide for computer-assisted primary diagnosis.

Mechanisms of Activation and Subunit Release in Ca2+/Calmodulin-Dependent Protein Kinase II.

Calcium/calmodulin-dependent protein kinase II is an enzyme involved in many different functions, including the so-called long-term potentiation, a mechanism that strengthens synapses in a persistent mode and is believed to be a basic cellular mechanism for memory formation. Here we study the conformational changes of the enzyme due to phosphorylation of some key residues that are believed to drive the transition from an inhibited to an active state; it is this active state the one associated with long-term potentiation. We found that the conformational changes could be explained in terms of three charged regions in the three main subdomains of the enzyme: the hub, linker, and kinase. The role of phosphorylation is to change the charge relation between them, turning on and off their interactions and switching between an attractive state (nonphosphorylated or inhibited) and a not attractive one (phosphorylated or active). We also show that phosphorylated subunits become less stable, and this could favor their release from the multimer, as has been already observed experimentally.

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble.

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

WNT5A inhibits hepatocyte proliferation and concludes ß-catenin signaling in liver regeneration.

Activation of Wnt/ß-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased ß-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in ß-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of ß-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses ß-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes.

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.

Early stages of beta2-microglobulin aggregation and the inhibiting action of alphaB-crystallin.

Static and dynamic light scattering experiments on extremely clean (nanofiltered) samples of the well-known amyloidogenic protein beta2-microglobulin (R3Abeta2m and WTbeta2m) evidence the self-assembly of early aggregates showing unexpected features. Further, we find that alphaB-crystallin effectively inhibits aggregation of beta2m in a far less than stoichiometric proportion, from 1:60 alphaB-crystallin monomer to beta2m monomer ratio, down to at least a 1:2 x 10(3) alphaB-crystallin oligomerto beta2m monomer ratio. Therefore, inhibition of the early stage of beta2m aggregation by alphaB-crystallin does not necessarily require a mechanicistic chaperon-like action implying one-to-one binding. This highlights the role of the free energy landscape of the system and of related modifications of solute-solvent thermodynamics caused by co-solutes, in agreement with recent work from our and other laboratories.

Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering.

To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein; disordered structures add as the pD decreases. Moreover, beta-aggregates seem to contribute only to oligomers formation, as showed by the good correlation between kinetics of scattering intensity and IR absorption intensity. These results indicate for BSA a general mechanism of aggregation composed by partial unfolding of the tertiary structure and by the decrease of alpha-helix and random coil contents in favor of beta-sheet aggregates. This mechanism strictly depends on pD and gives rise to almost two distinct types of macromolecular aggregates.