Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.

A Multiwavelength Study of the Intracluster Medium and the Characterization of the Multiwavelength Sub/millimeter Inductance Camera

The first part of this thesis combines Bolocam observations of the thermal Sunyaev-Zel’dovich (SZ) effect at 140 GHz with X-ray observations from Chandra, strong lensing data from the Hubble Space Telescope (HST), and weak lensing data from HST and Subaru to constrain parametric models for the distribution of dark and baryonic matter in a sample of six massive, dynamically relaxed galaxy clusters. For five of the six clusters, the full multiwavelength dataset is well described by a relatively simple model that assumes spherical symmetry, hydrostatic equilibrium, and entirely thermal pressure support. The multiwavelength analysis yields considerably better constraints on the total mass and concentration compared to analysis of any one dataset individually. The subsample of five galaxy clusters is used to place an upper limit on the fraction of pressure support in the intracluster medium (ICM) due to nonthermal processes, such as turbulent and bulk flow of the gas. We constrain the nonthermal pressure fraction at r500c to be less than 0.11 at 95% confidence, where r500c refers to radius at which the average enclosed density is 500 times the critical density of the Universe. This is in tension with state-of-the-art hydrodynamical simulations, which predict a nonthermal pressure fraction of approximately 0.25 at r500c for the clusters in this sample.

The second part of this thesis focuses on the characterization of the Multiwavelength Sub/millimeter Inductance Camera (MUSIC), a photometric imaging camera that was commissioned at the Caltech Submillimeter Observatory (CSO) in 2012. MUSIC is designed to have a 14 arcminute, diffraction-limited field of view populated with 576 spatial pixels that are simultaneously sensitive to four bands at 150, 220, 290, and 350 GHz. It is well-suited for studies of dusty star forming galaxies, galaxy clusters via the SZ Effect, and galactic star formation. MUSIC employs a number of novel detector technologies: broadband phased-arrays of slot dipole antennas for beam formation, on-chip lumped element filters for band definition, and Microwave Kinetic Inductance Detectors (MKIDs) for transduction of incoming light to electric signal. MKIDs are superconducting micro-resonators coupled to a feedline. Incoming light breaks apart Cooper pairs in the superconductor, causing a change in the quality factor and frequency of the resonator. This is read out as amplitude and phase modulation of a microwave probe signal centered on the resonant frequency. By tuning each resonator to a slightly different frequency and sending out a superposition of probe signals, hundreds of detectors can be read out on a single feedline. This natural capability for large scale, frequency domain multiplexing combined with relatively simple fabrication makes MKIDs a promising low temperature detector for future kilopixel sub/millimeter instruments. There is also considerable interest in using MKIDs for optical through near-infrared spectrophotometry due to their fast microsecond response time and modest energy resolution. In order to optimize the MKID design to obtain suitable performance for any particular application, it is critical to have a well-understood physical model for the detectors and the sources of noise to which they are susceptible. MUSIC has collected many hours of on-sky data with over 1000 MKIDs. This work studies the performance of the detectors in the context of one such physical model. Chapter 2 describes the theoretical model for the responsivity and noise of MKIDs. Chapter 3 outlines the set of measurements used to calibrate this model for the MUSIC detectors. Chapter 4 presents the resulting estimates of the spectral response, optical efficiency, and on-sky loading. The measured detector response to Uranus is compared to the calibrated model prediction in order to determine how well the model describes the propagation of signal through the full instrument. Chapter 5 examines the noise present in the detector timestreams during recent science observations. Noise due to fluctuations in atmospheric emission dominate at long timescales (less than 0.5 Hz). Fluctuations in the amplitude and phase of the microwave probe signal due to the readout electronics contribute significant 1/f and drift-type noise at shorter timescales. The atmospheric noise is removed by creating a template for the fluctuations in atmospheric emission from weighted averages of the detector timestreams. The electronics noise is removed by using probe signals centered off-resonance to construct templates for the amplitude and phase fluctuations. The algorithms that perform the atmospheric and electronic noise removal are described. After removal, we find good agreement between the observed residual noise and our expectation for intrinsic detector noise over a significant fraction of the signal bandwidth.

Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

A Social and Economic Characterization of the U.S. Gulf of Mexico Recreational Shark Fishery

A mail survey of tournament shark anglers and party boat shark anglers was completed to examine their fishing activity, attitudes, trip expenditures, and consumer surplus. A sample of 700 shark anglers was selected from tournaments in the Gulf of Mexico during 1990, and a sample of party boat shark anglers was drawn from Port Aransas, Tex., party boat anglers during the summer of 1991. A response rate of 58% (excluding nondeliverables) was obtained from tournament anglers. The sample of party boat shark anglers was too small to provide useful results. Tournament shark anglers reported fishing an average of 58 days per year and targeted sharks and other large marine species. Tournaments occupy a small portion of their fishing effort. If this group of anglers were not able to fish for sharks, one-third indicated no other species would be an acceptable substitute, while others were willing to substitute other large marine species. Shark trip expenditures averaged $197 per trip with a consumer surplus of $111 per trip. Based on MRFSS estimates of the number of shark fishing trips, we estimate a total of $43,355,000 was spent by shark anglers in the Gulf of Mexico with a consumer surplus of $23,865,000 for a total gross value of the shark fishery of $66,220,000. MRFSS estimates of the number of sharks landed indicate an equivalent use value of $183 per shark.

Characterization of the temperature sensitivity of gain and recombination mechanisms in 1.3-μm AlGaInAs MQW lasers

The potential of 1.3-μm AlGaInAs multiple quantum-well (MQW) laser diodes for uncooled operation in high-speed optical communication systems is experimentally evaluated by characterizing the temperature dependence of key parameters such as the threshold current, transparency current density, optical gain and carrier lifetime. Detailed measurements performed in the 20°C-100°C temperature range indicate a localized T0 value of 68 K at 98°C for a device with a 2.8μm ridge width and 700-μm cavity length. The transparency current density is measured for temperatures from 20°C to 60°C and found to increase at a rate of 7.7 A·cm -2 · °C-1. Optical gain characterizations show that the peak modal gain at threshold is independent of temperature, whereas the differential gain decreases linearly with temperature at a rate of 3 × 10-4 A-1·°C-1. The differential carrier lifetime is determined from electrical impedance measurements and found to decrease with temperature. From the measured carrier lifetime we derive the monomolecular (A), radiative (B), and nonradiative Auger (C) recombination coefficients and determine their temperature dependence in the 20 °C-80 °C range. Our study shows that A is temperature independent, B decreases with temperature, and C exhibits a less pronounced increase with temperature. The experimental observations are discussed and compared with theoretical predictions and measurements performed on other material systems. © 2005 IEEE.

Characterization of the U.S. Gulf of Mexico and South Atlantic penaeid and rock shrimp fisheries based on observer data

In July 2007, a mandatory Federal observer program was implemented to characterize the U.S. Gulf of Mexico penaeid shrimp (Farfantepenaeus aztecus, F. duorarum, and Litopenaeus setiferus) fishery. In June 2008, the program expanded to include the South Atlantic penaeid and rock shrimp, Sicyonia spp., fisheries. Data collected from 10,206 tows during 5,197 sea days of observations were analyzed by geographical area and target species. The majority of tows (~70%) sampled were off the coasts of Texas and Louisiana. Based on total hours towed, the highest concentrated effort occurred off South Texas and southwestern Florida. Gear information, such as net characteristics, bycatch reduction devices, and turtle excluder devices were fairly consistent among areas and target species. By species categories, finfish comprised the majority (≥57%) of the catch composition in the Gulf of Mexico and South Atlantic penaeid shrimp fisheries, while in the South Atlantic rock shrimp fishery the largest component (41%) was rock shrimp. Bycatch to shrimp ratios were lower than reported in previous studies for the Gulf of Mexico penaeid shrimp fishery. These decreased ratios may be attributed to several factors, notably decreased shrimp effort and higher shrimp catch per unit of effort (CPUE) in recent years. CPUE density surface plots for several species of interest illustrated spatial differences in distribution. Hot Spot Analyses for shrimp (penaeid and rock) and bycatch species identified areas with significant clustering of high or low CPUE values. Spatial and temporal distribution of protected species interactions were documented.

An ecological characterization of the Stellwagen Bank National Marine Sanctuary Region: oceanographic, biogeographic, and contaminants assessment

The mission of NOAA’s National Marine Sanctuary Program (NMSP) is to serve as the trustee for a system of marine protected areas, to conserve, protect, and enhance their biodiversity, ecological integrity, and cultural legacy while facilitating compatible uses. Since 1972, thirteen National Marine Sanctuaries, representing a wide variety of ocean environments, have been established, each with management goals tuned to their unique diversity. Extending from Cape Ann to Cape Cod across the mouth of Massachusetts Bay, Stellwagen Bank National Marine Sanctuary (NMS) encompasses 2,181 square kilometers of highly productive, diverse, and culturally unique Federal waters. As a result of its varied seafloor topography, oceanographic conditions, and high primary productivity, Stellwagen Bank NMS is utilized by diverse assemblages of seabirds, marine mammals, invertebrates, and fish species, as well as containing a number of maritime heritage resources. Furthermore, it is a region of cultural significance, highlighted by the recent discovery of several historic shipwrecks. Officially designated in 1992, Stellwagen Bank became the Nation’s twelfth National Marine Sanctuary in order to protect these and other unique biological, geological, oceanographic, and cultural features of the region. The Stellwagen Bank NMS is in the midst of its first management plan review since designation. The management plan review process, required by law, is designed to evaluate, enhance, and guide the development of future research efforts, education and outreach, and the management approaches used by Sanctuaries. Given the ecological and physical complexity of Stellwagen Bank NMS, burgeoning anthropogenic impacts to the region, and competing human and biological uses, the review process was challenged to assimilate and analyze the wealth of existing scientific knowledge in a framework which could enhance management decision-making. Unquestionably, the Gulf of Maine, Massachusetts Bay, and Stellwagen Bank-proper are extremely well studied systems, and in many regards, the scientific information available greatly exceeds that which is available for other Sanctuaries. However, the propensity of scientific information reinforces the need to utilize a comprehensive analytical approach to synthesize and explore linkages between disparate information on physical, biological, and chemical processes, while identifying topics needing further study. Given this requirement, a partnership was established between NOAA’s National Marine Sanctuary Program (NMSP) and the National Centers for Coastal Ocean Science (NCCOS) so as to leverage existing NOAA technical expertise to assist the Sanctuary in developing additional ecological assessment products which would benefit the management plan review process.