Jimmy El Hokayem

Label-Free Quantitative LC–MS Proteomics of Alzheimer’s Disease and Normally Aged Human Brains

Quantitative proteomics analysis of cortical samples of 10 Alzheimers disease (AD) brains versus 10 normally aged brains was performed by following the accurate mass and time tag (AMT) approach with the high resolution LTQ Orbitrap mass spectrometer. More than 1400 proteins were identified and quantitated. A conservative approach of selecting only the consensus results of four normalization methods was suggested and used. A total of 197 proteins were shown to be significantly differentially abundant (p-values <0.05, corrected for multiplicity of testing) in AD versus control brain samples. Thirty-seven of these proteins were reported as differentially abundant or modified in AD in previous proteomics and transcriptomics publications. The rest to the best of our knowledge are new. Mapping of the discovered proteins with bioinformatic tools revealed significant enrichment with differentially abundant proteins of pathways and processes known to be important in AD, including signal transduction, regulation of protein phosphorylation, immune response, cytoskeleton organization, lipid metabolism, energy production, and cell death.

FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures

In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200–1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001–1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.

E6AP in the Brain: One Protein, Dual Function, Multiple Diseases

E6-Associated Protein (E6AP), the founding member of the HECT (Homologus to E6AP C terminus) family of ubiquitin ligases, has been gaining increased attention from the scientific community. In addition to its ubiquitin ligase function, our laboratory has also identified steroid hormone receptor transcriptional coactivation as yet another essential function of this protein. Furthermore, it has been established that E6AP has a role in numerous diseases including cancers and neurological syndromes. In this review, we delineate genetic and biochemical knowledge of E6AP and we focus on its role in the pathobiology of neuro-developmental and neuro-aging diseases; bringing to light important gaps of knowledge related to the involvement of its well-studied ligase function versus the much less studied nuclear receptor transcriptional coactivation function in the pathogenesis of these diseases. Tackling these gaps of knowledge could reveal novel possible neuro-pathobiological mechanisms and present crucial information for the design of effective treatment modalities for devastating CNS diseases.

Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner.

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi‐protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non‐overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2–Sos1 and Grb2–Gab1 binary signaling complexes in concert in lieu of a composite Sos1–Grb2–Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2–Sos1 and Grb2–Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. Copyright

Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts

Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

Blood Derived Induced Pluripotent Stem Cells (iPSCs): Benefits, Challenges and the Road Ahead.

Since the creation of induced Pluripotent Stem Cells (iPSCs) ten years ago, hundreds of publications have demonstrated their considerable impact on disease modeling and therapy. In this commentary, we will summarize key milestones, benefits and challenges in the iPSC field. Furthermore, we will highlight blood as an effective and easily accessible source for patient-specific iPSCs derivation in the context of work done in our laboratory and others.

E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma

E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

Convergent Pathways in Idiopathic Autism Revealed by Time Course Transcriptomic Analysis of Patient-Derived Neurons

Potentially pathogenic alterations have been identified in individuals with autism spectrum disorders (ASDs) within a variety of key neurodevelopment genes. While this hints at a common ASD molecular etiology, gaps persist in our understanding of the neurodevelopmental mechanisms impacted by genetic variants enriched in ASD patients. Induced pluripotent stem cells (iPSCs) can model neurodevelopment in vitro, permitting the characterization of pathogenic mechanisms that manifest during corticogenesis. Taking this approach, we examined the transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls over a 135-day course of neuronal differentiation. Our data show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. This study reveals important and functionally validated insights into common processes altered in early neuronal development and corticogenesis and may contribute to ASD pathogenesis.

Loss of Angelman Syndrome Protein E6AP Disrupts a Novel Antagonistic Estrogen-Retinoic Acid Transcriptional Crosstalk in Neurons

Angelman syndrome (AS) is a complex genetic disorder that affects the nervous system. AS affects an estimated 1 in 12,000 to 20,000 individuals. Characteristic features of AS includes developmental delay or intellectual disability, severe speech impairment, seizures, small head size (microcephaly), and problems with movement and balance (ataxia). AS individuals usually have microdeletion of the maternal copy of 15q11.2–15q13 region of chromosome 15. The E6-associated protein (E6AP, an E3 ubiquitin protein ligase enzyme) is encoded by the gene UBE3A, which is located in this region, and it has been shown that deregulation of E6AP gives rise to AS and neuropathology of autism spectrum disorders (ASDs) (e.g., autism and Rett syndromes). We have shown that E6AP also acts as a coactivator of the estrogen receptor (ER). ER is a ligand-induced transcription factor that exerts potent and wide-ranging effects on the developing brain. Furthermore, the expression pattern of ER in the brain overlaps with that of E6AP. Up till now, all the published studies have examined the role of the ubiquitin-protein ligase activity of E6AP in the development of AS, and it is not known what role the newly discovered coactivation functions of E6AP and ER plays in the pathology of AS. Here, we demonstrate that E6AP and ER co-immunoprecipitate and are in the same protein complex in neuronal cells (Neuro2a). In addition, both colocalize in nuclear and cytoplasmic compartments of the mouse hippocampal neurons and Neuro2a cells. Moreover, we identified a novel E6AP and ER direct transcriptional regulation of a gene Cyp26b1 known to be involved in learning and memory processes. This transcriptional regulation involves recruitment of E6AP and ER to a newly discovered functional estrogen response element (ERE) located at the Cyp26b1 gene promoter and is associated with transcription permissive epigenetic events leading to increase of active transcription of the gene in neurons upon estrogen treatment. This novel transcriptional regulation was also validated in the AS mouse model where E6AP expression is abrogated in the mouse brain. In fact, Cyp26b1 expression is decreased by 31% in AS mice versus age-matched control (Ctrl) mice hippocampi. Also, retinoic acid transcriptional signaling was shown to be amplified as evidenced by specific increased Rarβ and decreased Erbb4 mRNA expression in AS mice versus Ctrl mice hippocampi. These transcript level changes were also supported by the same trend of changes at the protein level. Collectively, our data present a proof of principle that the transcriptional coactivation function of E6AP may have a crucial role in the pathobiology of AS. This function, yet to be thoroughly investigated, reveals the possibility of harnessing the antagonistic estrogen-retinoic acid transcriptional signaling crosstalk and potentially other unknown effectors for the investigation of important possible targets as putative novel treatment modalities and venues for reversing neurological manifestations in AS and related syndromes like ASDs.

Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells

The tumor necrosis factor receptor-associated factors (TRAFs) have been classically described as adaptor proteins that function as solely cytosolic signaling intermediates for the TNF receptor superfamily, Toll-like receptors (TLRs), NOD, like receptors (NLRs), cytokine receptors, and others. In this study, we show for the first time that TRAFs are present within the cytoplasm and nucleus of Neuro2a cells and primary cortical neurons, and that TRAF2 and TRAF3 translocate into the nucleus within minutes of CD40L stimulation. Analysis of the transcriptional regulatory potential of TRAFs by luciferase assay revealed that each of the TRAFs differentially functions as a transcriptional activator or repressor in a cell-specific manner. Interestingly, ChIP-qPCR data demonstrate that TRAFs 2/3, p65, and pRNAPol II form part of a transcriptional complex on the Icam-1 gene promoter upon CD40L stimulation. We further determined that TRAF2 recruitment to the nucleus is critical for the ubiquitination of H2b, a transcription permissive epigenetic modification. Our findings demonstrate for the first time that TRAFs 2/3 participate in the formation of a CD40L-induced transcriptional complex in neuronal cells.