Faculty Bibliography

Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details

In addition to serving as the entry point for newly translated polypeptides making their way through the secretory pathway, the endoplasmic reticulum (ER) also synthesizes many lipid components of the entire endomembrane system. A report published in this issue implicates a signaling pathway known to respond to ER unfolded protein load in the control of phospholipid biosynthesis by the organelle (Sriburi et al., 2004). The reasonable notion that demand for ER membrane is integrated with protein processing capacity was initially suggested by genetic analysis of yeast. The new data lend direct support for this idea and imply interesting mechanistic possibilities for how this coupling develops

Our purpose was to determine the clinical relevance of the detection of circulating tumor cells (CTCs) expressing urothelial and epithelial markers in bladder cancer patients. Sixty-two patients who presented to Memorial Sloan-Kettering Cancer Center between July 2000 and September 2001 were studied. Peripheral blood was tested by nested RT-PCR assay for uroplakins (UPs) Ia, Ib, II and III as well as for epidermal growth factor receptor (EGFR). We determined the sensitivity and specificity of each individual marker and the combinations of UPIa/UPII and UPIb/UPIII. The latter strategy was based on our data, which showed that UPIa and UPIb form heterodimers with UPII and UPIII, respectively. Forty patients had clinically advanced bladder cancer and 22 had no evidence of disease at the time of assay. Eight of the 22 patients recurred during the follow-up period. All 8 patients were positive at presentation for UPIa/UPII. The combination of UPIa/UPII provided the best sensitivity (75%) of detecting CTCs, with a specificity of 50%. The combination of UPIb/UPIII was the most specific (79%) but had modest sensitivity (31%). Detection of EGFR-positive cells alone and in combination with UPs was inferior to that for UPIa/UPII. Combinations of urothelial markers are superior to single urothelial or epithelial markers in detecting CTCs in bladder cancer patients. Further efforts are under way to confirm the potential predictive value of these markers in a prospectively designed study of a larger cohort of patients.

Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH

The alpha-conotoxins MI and GI display stronger affinities for the alphagamma agonist site on the Torpedo californica electrocyte nicotinic acetylcholine receptor (ACHR) than for the alphadelta agonist site, while alpha-conotoxin SI binds with the same affinity to both sites. Prior studies reported that the arginine at position 9 on GI and the tyrosine at position 111 on the receptor gamma subunit were responsible for the stronger alphagamma affinities of GI and MI, respectively. This study was undertaken to determine if the alpha-conotoxin midchain cationic residues interact with Torpedo gammaY111. The findings show that lysine 10 on MI is responsible for the alphagamma selectivity of MI and confirm the previously reported importance of R9 on GI and on the SI analogue, SIP9R. The results also show that gammaY111 contributes substantially to the selective alphagamma high affinity of all three peptides. Double-mutant cycle analyses reveal that, in the alphagamma site, K10 on MI and R9 on SIP9R interact with the aromatic ring of gammaY111 to stabilize the high-affinity complex, while in contrast, R9 on GI does not. The substitution of Y for R at position 113 on the delta subunit converts the alphadelta site into a high-affinity site for MI, GI, and SIP9R through the interacting of deltaY113 with K10 on MI and with R9 on both GI and SIP9R. The overall data show that the residues in the two sites with which MI interacts, other than at gamma111/delta113, are either the same or similar enough to exert equivalent effects on MI, indicating that MI binds in the same orientation at the alphagamma and alphadelta sites. Similar findings show that SIP9R probably also binds in the same orientation at the wild-type alphagamma and alphadelta sites. The finding that R9 on GI interacts closely with deltaR113Y but not with gammaY111 means that GI binds in different orientations at the alphagamma and alphadelta sites. This report also discusses the molecular basis of the difference in the MI high-affinity sites on Torpedo and embryonic mouse muscle ACHRs.

Organismal physiology depends significantly on the proper assembly of extracellular matrix (ECM) macroaggregates that impart structural integrity to the connective tissue. Recent genetic studies in mice have unraveled unsuspected new functions of architectural matrix components in regulating signaling events that modulate patterning, morphogenesis, and growth of several organ systems. As a result, a new paradigm has emerged whereby tissue-specific organization of the ECM dictates not only the physical properties of the connective tissue, but also the ability of the matrix to direct a broad spectrum of cellular activities through the regulation of growth factor signaling. These observations pave the way to novel therapeutic approaches aimed at counteracting the deleterious consequences of perturbations of connective tissue homeostasis.

CNS synapses are complex sites of cell-cell communication. Identification and characterization of the protein components of synapses will lead to a better understanding of the mechanisms of neurotransmission and plasticity. We applied multidimensional protein identification technology (MudPIT) to purified, guanidine-solubilized postsynaptic fractions to identify novel synaptically localized molecules. We identified several actin-associated proteins known to regulate actin polymerization and control cell motility in nonneural cells that have not previously been associated with CNS synaptic function. One of these is lasp-1, an actin-associated LIM and SH3 domain-containing protein. We show that lasp-1 is strongly expressed by CNS neurons and is concentrated at synaptic sites. Overall, the preponderance of actin-associated proteins in postsynaptic density fractions, and specifically those involved in actin reorganization, suggests that there are many modes by which the state of synaptic F-actin polymerization and, hence, synaptic physiology are affected.

E2A transcription factors, E12 and E47, are important regulators of lymphocyte development. Notch signaling pathways have been shown to regulate E2A function by accelerating the degradation of E2A proteins through a mitogen-activated protein kinase-dependent and ubiquitin-mediated pathway. To further understand the mechanism underlying E2A ubiquitination and degradation, we conducted a yeast two-hybrid screen and identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as an E47 binding protein. Here, we show that CHIP associates with E2A proteins in vivo and that overexpression of CHIP induces E47 degradation in a phosphorylation-dependent manner. Conversely, knocking down CHIP with small interfering RNA alleviates Notch-induced E47 degradation. CHIP binds E47 through the E protein homology domains 2 and 3 (EHD2 and EHD3). This interaction between CHIP and E47 is independent of the U-box domain with E3 ubiquitin ligase activity but requires the chaperone binding tetratricopeptide repeats domain. The ability of CHIP to induce E47 ubiquitination and degradation correlates with its ability to bind E47. We propose that CHIP, together with its partner Hsc70, forms a preubiquitination complex (PUC) with E47 and Skp2, thus facilitating the interaction between E47 and Skp2. CHIP also associates with Cul1, which introduces PUC to the SCF E3 ligase complex, responsible for E47 ubiquitination. Therefore, CHIP plays a crucial role in the ubiquitination and degradation of E2A proteins.

Transporting epithelia posed formidable conundrums right from the moment that Du Bois Raymond discovered their asymmetric behavior, a century and a half ago. It took a century and a half to start unraveling the mechanisms of occluding junctions and polarity, but we now face another puzzle: lest its cells died in minutes, the first high metazoa (i.e., higher than a sponge) needed a transporting epithelium, but a transporting epithelium is an incredibly improbable combination of occluding junctions and cell polarity. How could these coincide in the same individual organism and within minutes? We review occluding junctions (tight and septate) as well as the polarized distribution of Na(+)-K(+)-ATPase both at the molecular and the cell level. Junctions and polarity depend on hosts of molecular species and cellular processes, which are briefly reviewed whenever they are suspected to have played a role in the dawn of epithelia and metazoan. We come to the conclusion that most of the molecules needed were already present in early protozoan and discuss a few plausible alternatives to solve the riddle described above.