Faculty Bibliography

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (Riam). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for Riam we show that the Rap1/Riam module is not required for T or B cell development but is essential for efficient adhesion to ICAM-1 and VCAM-1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in Riam deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lympyhocyte trafficking was accompanied by defective humoral immunity to T cell-dependent antigens. Platelet function was intact in Riam deficient animals. These in vivo results confirm a role for Riam in the regulation of some, but not all, leukocyte integrins and suggest that Riam-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.

The immune system recognizes protein antigens expressed in transformed cells evidenced by accumulation of antigen-specific T cells in tumor and tumor draining lymph nodes. However, despite demonstrable immune response, cancers grow progressively suggesting that priming of antitumor immunity is insufficiently vigorous or that antitumor immunity is suppressed, or both. Compared to virus infection, antitumor T cells are low abundance that likely contributes to tumor escape and enhancement of priming is a long-sought goal of experimental vaccination therapy. Furthermore, patient treatment with antigen-specific T cells can in some cases overcome deficient priming and cause tumor regression supporting the notion that low numbers of T cells permits tumor outgrowth. However, tumor-induced suppression of antitumor immune response is now recognized as a significant factor contributing to cancer growth and reversal of the inhibitory influences within the tumor microenvironment is a major research objective. Multiple cell types and factors can inhibit T cell functions in tumors and may be grouped in two general classes: T cell intrinsic and T cell extrinsic. T cell intrinsic factors are exemplified by T cell expression of cell surface inhibitory signaling receptors that, after contact with cells expressing a cognate ligand, inactivate proximal T Cell Receptor-mediated signal transduction therein rendering T cells dysfunctional. T cell extrinsic factors are more diverse in nature and are produced by tumors and various non-tumor cells in the tumor microenvironment including: regulatory T cells, B cells, myeloid-derived cells, cancer associated fibroblasts, pericytes, mast cells, or vascular endothelia. These include proteins secreted by tumor or stromal cells (some which may be retained in the extracellular matrix), highly reactive soluble oxygen and nitrogen species, cytokines, chemokines, gangliosides, and toxic metabolites. These factors may restrict T cell entrance into the tumor parenchyma, cause inactivation of effector phase T cell functions, or induce T cell apoptosis ultimately causing diminished cancer elimination. Here, we review the contributions of inhibitory factors to tumor T cell dysfunction leading to tumor escape.

Stress triggered neuropsychiatric disorders are a serious societal problem. Prophylactic treatment or early intervention has great potential in increasing resilience to traumatic stress and reducing its harmful impact. Previously, we demonstrated proof of concept that intranasal administration of neuropeptide Y (NPY) or the melanocortin receptor four (MC4R) antagonist, HS014, prior to single prolonged stress (SPS) rodent post-traumatic stress disorder (PTSD) model, can prevent or attenuate many PTSD associated impairments. Here, we compare effects of NPY or HS014 given 30min before or immediately after SPS stressors on development of anxiety, depressive-like behavior and associated biochemical abnormalities. SPS triggered anxiety on elevated plus maze (EPM) was reduced by intranasal administration of 100mug NPY and to even greater extent HS014 (3.5ng or 100mug). The SPS-elicited depressive-like behavior on forced swim test was prevented with 100mug NPY or the high dose HS014. Combined administration of low HS014 and NPY, ineffective by themselves, prevented development of depressive-like behavior. Reductions in stress triggered activation of locus coeruleus/noradrenergic system and HPA axis were observed with both HS014 and NPY. In contrast to NPY which has been showed earlier, infusion of HS014 immediately after SPS did not prevent the development of anxiogenic behavior on EPM. However, HS014 given after SPS stressors effectively even at very low dose, prevented development of depressive-like behavior. Thus, both MC4R antagonist and NPY, alone or combined, have potential for prophylactic treatment against traumatic stress triggered anxiety or depressive-like behaviors, while NPY has more widespread potential for early intervention.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Although prokaryotes are usually classified using molecular phylogenies instead of phenotypes after the advent of gene sequencing, neither of these methods is satisfactory because the phenotypes cannot explain the molecular trees and the trees do not fit the phenotypes. This scientific crisis still exists and the profound disconnection between these two pillars of evolutionary biology-genotypes and phenotypes-grows larger. We use rings and a genomic form of goods thinking to resolve this conundrum (McInerney JO, Cummins C, Haggerty L. 2011. Goods thinking vs. tree thinking. Mobile Genet Elements. 1:304-308; Nelson-Sathi S, et al. 2015. Origins of major archaeal clades correspond to gene acquisitions from bacteria. Nature 517:77-80). The Proteobacteria is the most speciose prokaryotic phylum known. It is an ideal phylogenetic model for reconstructing Earth's evolutionary history. It contains diverse free living, pathogenic, photosynthetic, sulfur metabolizing, and symbiotic species. Due to its large number of species (Whitman WB, Coleman DC, Wiebe WJ. 1998. Prokaryotes: the unseen majority. Proc Nat Acad Sci U S A. 95:6578-6583) it was initially expected to provide strong phylogenetic support for a proteobacterial tree of life. But despite its many species, sequence-based tree analyses are unable to resolve its topology. Here we develop new rooted ring analyses and study proteobacterial evolution. Using protein family data and new genome-based outgroup rooting procedures, we reconstruct the complex evolutionary history of the proteobacterial rings (combinations of tree-like divergences and endosymbiotic-like convergences). We identify and map the origins of major gene flows within the rooted proteobacterial rings (P < 3.6 x 10(-6)) and find that the evolution of the "Alpha-," "Beta-," and "Gammaproteobacteria" is represented by a unique set of rings. Using new techniques presented here we also root these rings using outgroups. We also map the independent flows of genes involved in DNA-, RNA-, ATP-, and membrane- related processes within the Proteobacteria and thereby demonstrate that these large gene flows are consistent with endosymbioses (P < 3.6 x 10(-9)). Our analyses illustrate what it means to find that a gene is present, or absent, within a gene flow, and thereby clarify the origin of the apparent conflicts between genotypes and phenotypes. Here we identify the gene flows that introduced photosynthesis into the Alpha-, Beta-, and Gammaproteobacteria from the common ancestor of the Actinobacteria and the Firmicutes. Our results also explain why rooted rings, unlike trees, are consistent with the observed genotypic and phenotypic relationships observed among the various proteobacterial classes. We find that ring phylogenies can explain the genotypes and the phenotypes of biological processes within large and complex groups like the Proteobacteria.

Several systems-level datasets designed to dissect host-pathogen interactions during influenza A infection have been reported. However, apparent discordance among these data has hampered their full utility toward advancing mechanistic and therapeutic knowledge. To collectively reconcile these datasets, we performed a meta-analysis of data from eight published RNAi screens and integrated these data with three protein interaction datasets, including one generated within the context of this study. Further integration of these data with global virus-host interaction analyses revealed a functionally validated biochemical landscape of the influenza-host interface, which can be queried through a simplified and customizable web portal (http://www.metascape.org/IAV). Follow-up studies revealed that the putative ubiquitin ligase UBR4 associates with the viral M2 protein and promotes apical transport of viral proteins. Taken together, the integrative analysis of influenza OMICs datasets illuminates a viral-host network of high-confidence human proteins that are essential for influenza A virus replication.

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Delta14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Delta14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials.

The assessment, diagnosis, and management of fractures, particularly fractures that exhibit delayed healing, present considerable unique challenges to both patients and physicians. Fracture healing results from a complex series of biochemical events that may produce complete restoration of the anatomic and biochemical properties of the original osseous tissue. Fracture healing requires appropriate reduction, mechanical stability, and adequate vascularity to the fracture site; compromise of one of these elements may lead to delayed healing or nonunion. The patient's history, physical examination, and findings based on radiographs or other imaging modalities allow for assessment and characterization of the progression of healing. If nonunion is recognized, it is important for the clinician to understand the current treatment options that are available to optimize healing. Physical stimulation therapies include electromagnetic stimulation and low-intensity pulsed ultrasonography. Osteogenic factors used locally to promote fracture healing include autologous bone marrow and peptide signaling molecules such as platelet-derived growth factors, fibroblast growth factors, and bone morphogenetic proteins. Systemic biological protein such as parathyroid hormone and factors that target the Wnt family of signaling molecules offers promising data regarding its abilities to promote healing. Large segmental defects must be managed depending on the type and severity of the injury and may require treatment with bone grafts, induced membrane techniques, acute shortening, or distraction osteogenesis. A systematic approach in evaluating fracture union and an understanding of the modern methods to promote fracture healing will allow clinicians to significantly improve the treatment of patients with these injuries.