The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum

Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.     

Power-limited contraction dynamics of Vorticella convallaria: an ultrafast biological spring

Vorticella convallaria is one of the fastest and most powerful cellular machines. The cell body is attached to a substrate by a slender stalk containing a polymeric structure—the spasmoneme. Helical coiling of the stalk results from rapid contraction of the spasmoneme, an event mediated by calcium binding to a negatively charged polymeric backbone. We use high speed imaging to measure the contraction velocity as a function of the viscosity of the external environment and find that the maximum velocity scales inversely with the square root of the viscosity. This can be explained if the rate of contraction is ultimately limited by the power delivered by the actively contracting spasmoneme. Microscopically, this scenario would arise if the mechanochemical wave that propagates along the spasmoneme is faster than the rate at which the cell body can respond due to its large hydrodynamic resistance. We corroborate this by using beads as markers on the stalk and find that the contraction starts at the cell body and proceeds down the stalk at a speed that exceeds the velocity of the cell body.     

Program review. Challenges and opportunities for training the next generation of biophysicists: perspectives of the directors of the Molecular Biophysics Training Program at Northwestern University

Molecular biophysics is a broad, diverse, and dynamic field that has presented a variety of unique challenges and opportunities for training future generations of investigators. Having been or currently being intimately associated with the Molecular Biophysics Training Program at Northwestern, we present our perspectives on various issues that we have encountered over the years. We propose no cookie-cutter solutions, as there is no consensus on what constitutes the "ideal" program. However, there is uniformity in opinion on some key issues that might be useful to those interested in establishing a biophysics training program.     

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.     

Cysteine pKa depression by a protonated glutamic acid in human DJ-1

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K a of 5.4 +/- 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p K a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p K a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p K a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Non-linear control of continuous bioreactors

Control of bioreactors has achieved importance in the recent years. This may be due to the fact that they are difficult to control which may be attributed to its nonlinear dynamic behavior. The model parameters of the bioreactor also vary in an unpredictable manner. The complexity of the biochemical processes inhibits the accurate modeling and also the lack of suitable sensors make the process state difficult to characterize. Considerable emphasis has been placed on the control of fed-batch fermentors because of their prevalence in industries. However, when production of biomass is to be optimized, continuous operation is desirable. Several procedures are available for the nonlinear control of processes, viz., differential geometric approach, internal model control approach, reference synthesis technique, predictive control design, etc., but the major disadvantage of these approaches is the computational time required to perform the prediction optimization. In this study, a nonlinear controller based on a polynomial discrete time model (NARMAX) is evaluated for its performance on a fermentor. It can be shown that a nonlinear self-tuning controller based on NARMAX model can be extended to the control of fermentors. The response is smooth for both load and setpoint changes even when process parameters are assumed to be zero and uncertainty in parameters are present and in the presence of controller constraints. The control action can be made more or less robust by changing the design parameters appropriately. Therefore, nonlinear self-tuning controller is suitable for control of industrial processes.