DRAK2 contributes to type 1 diabetes by negatively regulating IL-2 sensitivity to alter regulatory T cell development.

Drak2-deficient (Drak2-/-) mice are resistant to multiple models of autoimmunity yet effectively eliminate pathogens and tumors. Thus, DRAK2 represents a potential target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Here, we demonstrate that resistance to T1D in non-obese diabetic (NOD) mice is due to the absence of Drak2 in T cells and requires the presence of regulatory T cells (Tregs). Contrary to previous hypotheses, we show that DRAK2 does not limit TCR signaling. Rather, DRAK2 regulates IL-2 signaling by inhibiting STAT5A phosphorylation. We further demonstrate that enhanced sensitivity to IL-2 in the absence of Drak2 augments thymic Treg development. Overall, our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic Treg development and by impacting the sensitivity of conventional T cells to Treg-mediated suppression.

Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity.

Infection and vaccination repeatedly expose individuals to antigens that are conserved between influenza virus subtypes. Nevertheless, antibodies recognizing variable influenza epitopes greatly outnumber antibodies reactive against conserved epitopes. Elucidating factors contributing to the paucity of broadly reactive influenza antibodies remains a major obstacle for developing a universal influenza vaccine. Here, we report that inducing broadly reactive influenza antibodies increases autoreactive antibodies in humans and mice and exacerbates disease in four distinct models of autoimmune disease. Importantly, transferring broadly reactive influenza antibodies augments disease in the presence of inflammation or autoimmune susceptibility. Further, broadly reactive influenza antibodies spontaneously arise in mice with defects in B cell tolerance. Together, these data suggest that self-tolerance mechanisms limit the prevalence of broadly reactive influenza antibodies, which can exacerbate disease in the context of additional risk factors.

Pre-existing humoral immunity to human common cold coronaviruses negatively impacts the protective SARS-CoV-2 antibody response.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

Characterization of a temporoparietal junction subtype of Alzheimer's disease.

Alzheimer's disease (AD) subtypes have been described according to genetics, neuropsychology, neuropathology, and neuroimaging. Thirty-one patients with clinically probable AD were selected based on perisylvian metabolic decrease on FDG-PET. They were compared to 25 patients with a typical pattern of decreased posterior metabolism. Tree-based machine learning was used on those 56 images to create a classifier that was subsequently applied to 207 Alzheimer's Disease Neuroimaging Initiative (ADNI) patients with AD. Machine learning was also used to discriminate between the two ADNI groups based on neuropsychological scores. Compared to AD patients with a typical precuneus metabolic decrease, the new subtype showed stronger hypometabolism in the temporoparietal junction. The classifier was able to distinguish the two groups in the ADNI population. Both groups could only be distinguished cognitively by Trail Making Test-A scores. This study further confirms that there is more than a typical metabolic pattern in probable AD with amnestic presentation.

Random Forests Based Group Importance Scores and Their Statistical Interpretation: Application for Alzheimer's Disease.

Machine learning approaches have been increasingly used in the neuroimaging field for the design of computer-aided diagnosis systems. In this paper, we focus on the ability of these methods to provide interpretable information about the brain regions that are the most informative about the disease or condition of interest. In particular, we investigate the benefit of group-based, instead of voxel-based, analyses in the context of Random Forests. Assuming a prior division of the voxels into non overlapping groups (defined by an atlas), we propose several procedures to derive group importances from individual voxel importances derived from Random Forests models. We then adapt several permutation schemes to turn group importance scores into more interpretable statistical scores that allow to determine the truly relevant groups in the importance rankings. The good behaviour of these methods is first assessed on artificial datasets. Then, they are applied on our own dataset of FDG-PET scans to identify the brain regions involved in the prognosis of Alzheimer's disease.

Extracellular Signal-Regulated Kinase Signaling in CD4-Expressing Cells Inhibits Osteochondromas.

Defects in cartilage homeostasis can give rise to various skeletal disorders including osteochondromas. Osteochondromas are benign bone tumors caused by excess accumulation of chondrocytes, the main cell type of cartilage. The extracellular signal-regulated kinase (ERK) pathway is a major signaling node that functions within chondrocytes to regulate their growth and differentiation. However, it is not known whether the ERK pathway in other cell types regulates cartilage homeostasis. We show here that mice with a germline deficiency of Erk1 and a conditional deletion of Erk2 in cells that express CD4, or expressed CD4 at one point in development, unexpectedly developed bone deformities. The bone lesions were due to neoplastic outgrowths of chondrocytes and disordered growth plates, similar to tumors observed in the human disease, osteochondromatosis. Chondrocyte accumulation was not due to deletion of Erk2 in the T cells. Rather, CD4cre was expressed in cell types other than T cells, including a small fraction of chondrocytes. Surprisingly, the removal of T cells accelerated osteochondroma formation and enhanced disease severity. These data show for the first time that T cells impact the growth of osteochondromas and describe a novel model to study cartilage homeostasis and osteochondroma formation.

The kinase mTOR modulates the antibody response to provide cross-protective immunity to lethal infection with influenza virus.

Highly pathogenic avian influenza viruses pose a continuing global threat. Current vaccines will not protect against newly evolved pandemic viruses. The creation of 'universal' vaccines has been unsuccessful because the immunological mechanisms that promote heterosubtypic immunity are incompletely defined. We found here that rapamycin, an immunosuppressive drug that inhibits the kinase mTOR, promoted cross-strain protection against lethal infection with influenza virus of various subtypes when administered during immunization with influenza virus subtype H3N2. Rapamycin reduced the formation of germinal centers and inhibited class switching in B cells, which yielded a unique repertoire of antibodies that mediated heterosubtypic protection. Our data established a requirement for the mTORC1 complex in B cell class switching and demonstrated that rapamycin skewed the antibody response away from high-affinity variant epitopes and targeted more conserved elements of hemagglutinin. Our findings have implications for the design of a vaccine against influenza virus.

Revisiting the role of the immunoproteasome in the activation of the canonical NF-κB pathway.

The discovery of NF-κB signaling pathways has greatly enhanced our understanding of inflammatory and immune responses. In the canonical NF-κB pathway, the proteasomal degradation of IκBα, an inhibitory protein of NF-κB, is widely accepted to be a key regulatory step. However, contradictory findings have been reported as to whether the immunoproteasome plays an obligatory role in the degradation of IκBα and activation of the canonical NF-κB pathway. Such results were obtained mainly using traditional gene deletion strategies. Here, we have revisited the involvement of the immunoproteasome in the canonical NF-κB pathway using small molecule inhibitors of the immunoproteasome, namely UK-101 and LKS01 targeting ß1i and ß5i, respectively. H23 and Panc-1 cancer cells were pretreated with UK-101, LKS01 or epoxomicin (a prototypic inhibitor targeting both the constitutive proteasome and immunoproteasome). We then examined whether these pretreatments lead to any defect in activating the canonical NF-κB pathway following TNFα exposure by monitoring the phosphorylation and degradation of IκBα, nuclear translocation of NF-κB proteins and DNA binding and transcriptional activity of NF-κB. Our results consistently indicated that there is no defect in activating the canonical NF-κB pathway following selective inhibition of the immunoproteasome catalytic subunits ß1i, ß5i or both using UK-101 and LKS01, in contrast to epoxomicin. In summary, our current results using chemical genetic approaches strongly support that the catalytic activity of the immunoproteasome subunits ß1i and ß5i is not required for canonical NF-κB activation in lung and pancreatic adenocarcinoma cell line models.

A bright approach to the immunoproteasome: development of LMP2/ß1i-specific imaging probes.

While the constitutive, 26S proteasome plays an important role in regulating many important cellular processes, a variant form known as the immunoproteasome is thought to primarily function in adaptive immune responses. However, recent studies indicate an association of immunoproteasomes with many physiological disorders such as cancer, neurodegenerative, and inflammatory diseases. Despite this, the detailed functions of the immunoproteasome remain poorly understood. Immunoproteasome-specific probes are essential to gain insight into immunoproteasome function. Here, we describe for the first time the development of cell-permeable activity-based fluorescent probes, UK101-Fluor and UK101-B660, which selectively target the catalytically active LMP2/ß1i subunit of the immunoproteasome. These probes facilitate rapid detection of the cellular localization of catalytically active immunoproteasomes in living cells, providing a valuable tool to analyze immunoproteasome functions. Additionally, as LMP2/ß1i may serve as a potential tumor biomarker, an LMP2/ß1i-targeting fluorescent imaging probe may be applicable to a rapid readout assay to determine tumor LMP2/ß1i levels.

Impact of linker length on the activity of PROTACs.

Conventional genetic approaches have provided a powerful tool in the study of proteins. However, these techniques often preclude selective manipulation of temporal and spatial protein functions, which is crucial for the investigation of dynamic cellular processes. To overcome these limitations, a small molecule-based novel technology termed "PROteolysis TArgeting ChimeraS (PROTACs)" has been developed, targeting proteins for degradation at the post-translational level. Despite the promising potential of PROTACs to serve as molecular probes of complex signaling pathways, their design has not been generalized for broad application. Here, we present the first generalized approach for PROTAC design by fine-tuning the distance between the two participating partner proteins, the E3 ubiquitin ligase and the target protein. As such, we took a chemical approach to create estrogen receptor (ER)-α targeting PROTACs with varying linker lengths and the loss of the ER in cultured cells was monitored via western blot and fluorometric analyses. We found a significant effect of chain length on PROTAC efficacy, and, in this case, the optimum distance between the E3 recognition motif and the ligand was a 16 atom chain length. The information gathered from this experiment may offer a generalizable PROTAC design strategy to further the expansion of the PROTAC toolbox, opening new possibilities for the broad application of the PROTAC strategy in the study of multiple signaling pathways.

Molecular basis of the selectivity of the immunoproteasome catalytic subunit LMP2-specific inhibitor revealed by molecular modeling and dynamics simulations.

Given that immunoproteasome inhibitors are currently being developed for a variety of potent therapeutic purposes, the unique specificity of an α',ß'-epoxyketone peptide (UK101) toward the LMP2 subunit of the immunoproteasome (analogous to ß5 subunit of the constitutive proteasome) has been investigated in this study for the first time by employing homology modeling, molecular docking, molecular dynamics simulation, and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations. On the basis of the simulated binding structures, the calculated binding free energies are in qualitative agreement with the corresponding experimental data, and the selectivity of UK101 is explained reasonably. The observed selectivity of UK101 for the LMP2 subunit is rationalized by the requirement for both a linear hydrocarbon chain at the N terminus and a bulky group at the C terminus of the inhibitor, because the LMP2 subunit has a much more favorable hydrophobic pocket interacting with the linear hydrocarbon chain, and the bulky group at the C terminus has a steric clash with the Tyr 169 in ß5 subunit. Finally, our results help to clarify why UK101 is specific to the LMP2 subunit of immunoproteasome, and this investigation should be valuable for rational design of more potent LMP2-specific inhibitors.

Jostling for position: optimizing linker location in the design of estrogen receptor-targeting PROTACs.

Estrogen receptor-alpha (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone-sensitive ER-positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof-of-concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER-targeting PROTACs are composed of an estradiol on one end and a hypoxia-inducing factor 1alpha (HIF-1alpha)-derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7alpha position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex).

Crystal structure of Baeyer-Villiger monooxygenase MtmOIV, the key enzyme of the mithramycin biosynthetic pathway .

Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV's structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9 A. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments identifies several residues that participate in cofactor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis.

Proteasome modulators: essential chemical genetic tools for understanding human diseases.

Primarily used for medicinal purposes in the past, biologically active small molecules have been increasingly employed to explore complex biological processes in the era of "chemical genetics". Since the contributions of this small molecule approach to biology have been extensive, we limit the focus of our review to the use of small-molecule modulators in the exciting field of proteasomal biology, one that has benefited significantly from a chemical genetics approach. Specifically, as the contributions of general inhibitors of proteasomal activity to the fields of cell biology and clinical oncology have been extensively discussed in several excellent reviews, we instead outline recent progress towards the development of novel, specific classes of proteasome modulators for studies of proteasomal biology and the types of proteasome inhibitors emerging as important new treatment options for cancer therapeutics.

LMP2-specific inhibitors: chemical genetic tools for proteasome biology.

The immunoproteasome, having been linked to neurodegenerative diseases and hematological cancers, has been shown to play an important role in MHC class I antigen presentation. However, its other pathophysiological functions are still not very well understood. This can be attributed mainly to a lack of appropriate molecular probes that can selectively modulate the immunoproteasome catalytic subunits. Herein, we report the development of molecular probes that selectively inhibit the major catalytic subunit, LMP2, of the immunoproteasome. We show that these compounds irreversibly modify the LMP2 subunit with high specificity. Importantly, LMP2-rich cancer cells compared to LMP2-deficient cancer cells are more sensitive to growth inhibition by the LMP2-specific inhibitor, implicating an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2.