Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17°C higher melting temperature. Its temperature optimum is 22–25°C higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a “corresponding state” regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation—phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Blind separation of auditory event-related brain responses into independent components

Averaged event-related potential (ERP) data recorded from the human scalp reveal electroencephalographic (EEG) activity that is reliably time-locked and phase-locked to experimental events. We report here the application of a method based on information theory that decomposes one or more ERPs recorded at multiple scalp sensors into a sum of components with fixed scalp distributions and sparsely activated, maximally independent time courses. Independent component analysis (ICA) decomposes ERP data into a number of components equal to the number of sensors. The derived components have distinct but not necessarily orthogonal scalp projections. Unlike dipole-fitting methods, the algorithm does not model the locations of their generators in the head. Unlike methods that remove second-order correlations, such as principal component analysis (PCA), ICA also minimizes higher-order dependencies. Applied to detected—and undetected—target ERPs from an auditory vigilance experiment, the algorithm derived ten components that decomposed each of the major response peaks into one or more ICA components with relatively simple scalp distributions. Three of these components were active only when the subject detected the targets, three other components only when the target went undetected, and one in both cases. Three additional components accounted for the steady-state brain response to a 39-Hz background click train. Major features of the decomposition proved robust across sessions and changes in sensor number and placement. This method of ERP analysis can be used to compare responses from multiple stimuli, task conditions, and subject states.

Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I− polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures

Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL’s affinity for unfolded proteins, and promotes degradation of certain polypeptides. Because the latter effects appeared larger at 20°C, we studied the influence of temperature on TF expression. Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42°C to 16°C and even rose in cells stored at 4°C. Upon temperature downshift from 37°C to 10°C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins. We therefore tested if TF expression might be important for viability at low temperatures. When stored at 4°C, E. coli lose viability at exponential rates. Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability. Although TF overproduction protected against cold, it reduced viability at 50°C, while TF deficiency enhanced viability at this temperature. By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4°C, which may explain why expression of hsps is suppressed in the cold. Thus, TF represents an example of an E. coli protein which protects cells against low temperatures. Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.

Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis

Relaxation of imprinting at the insulin-like growth factor II (IFG-II)/H19 locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of IFG-II imprinting, we determined the frequency of epigenetic change at the IFG-II/H19 locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of IFG-II imprinting and biallelic H19 methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among “normal” tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the IFG-II/H19 locus.

CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication

The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene. We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G1, after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins. These complexes are then triggered at the onset of S phase to commence DNA replication. Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication. In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G1/S phase transition, are dependent on one another for function. These data indicate that CDC45 functions in late G1 phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Gene Conversion of Major Histocompatibility Complex Genes in the Mouse Spermatogenesis is a Premeiotic Event

The molecular genetic mechanism of gene conversion in higher eukaryotes remains unknown. We find it of considerable interest to determine when during spermatogenesis gene conversion occurs. We have therefore purified pachytene spermatocytes and haploid spermatocytes from adult mice and analyzed these fractions for the presence of gene conversion products resulting from the transfer between the major histocompatibility complex class II genes Ebd and Abk in a polymerase chain reaction assay. We have further isolated spermatogenic cells from prepubescent mice and analyzed them for the presence of the same gene conversion products. We can detect gene conversion products in testis cells as early as in 8-d-old mice where the only existing spermatogenic cells are spermatogonia. The frequency of gene conversion products remains the same as the cells reach meiosis in 18-d-old mice, and is unchanged after meiosis is completed in haploid spermatocytes. Gene conversion of this specific fragment therefore appears to be a premeiotic event and, consequently, relies on genetic mechanisms other than normal meiotic recombination.