Faculty Bibliography

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFbeta signaling. TGFbeta is secreted from cells as a latent complex consisting of TGFbeta, the TGFbeta propeptide, and a molecule of latent TGFbeta binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFbeta levels, and has been hypothesized as an explanation for enhanced TGFbeta signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFbeta signaling in MFS might be due to defective interaction of latent TGFbeta complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFbeta complexes composed of LTBP-3 and TGFbeta contributes to aortic disease progression.

Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis.

The LTBPs (or latent transforming growth factor beta binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFbeta by covalently binding the TGFbeta propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFbeta precursor in the trans-golgi network but LAP and TGFbeta remain strongly bound through non-covalent interactions. LAP, TGFbeta, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFbeta latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFbeta activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFbeta regulation, TGFbeta-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.

During normal tissue development, the accumulation of unrepaired cellular and genomic damage can impair growth and ultimately leads to death. To preserve cellular integrity, cells employ a number of defense mechanisms including molecular checkpoints, during which development is halted while dedicated pathways attempt repair. This process is most critical in germline tissues where cellular damage directly threatens an organism's reproductive capacity and offspring viability. In the fruit fly, Drosophila melanogaster, germline development has been extensively studied for over a century and the breadth of our knowledge has flourished in the genomics age. Intriguingly, several peculiar phenomena that trigger catastrophic germline damage described decades ago, still endure only a partial understanding of the underlying molecular causes. A deeper reexamination using new molecular and genetic tools may greatly benefit our understanding of host system biology. Among these, and the focus of this concise review, are hybrid dysgenesis and an intragenomic conflict that pits the X and Y sex chromosomes against each other.

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.

DNA double-strand breaks (DSBs) disrupt the continuity of chromosomes and their repair by error-free mechanisms is essential to preserve genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism that involves alignment of microhomologous sequences internal to the broken ends before joining, and is associated with deletions and insertions that mark the original break site, as well as chromosome translocations. Whether MMEJ has a physiological role or is simply a back-up repair mechanism is a matter of debate. Here we review recent findings pertaining to the mechanism of MMEJ and discuss its role in normal and cancer cells.

Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability. Because these methods allow thousands of homologs to be examined in silico, they have the advantage of providing both speed and statistical power. Using these methods, we introduced, via mutation, amino acids from thermoadapted homologs into an exemplar mesophilic membrane protein, and demonstrated significantly increased thermostability while preserving protein activity.