The nuclear lamins A, B, and C are intermediate filament proteins that form a nuclear scaffold adjacent to the inner nuclear membrane in higher eukaryotes, providing structural support for the nucleus. In the past two decades it has become evident that the final step in the biogenesis of the mature lamin A from its precursor prelamin A by the zinc metalloprotease ZMPSTE24 plays a critical role in human health. Defects in prelamin A processing by ZMPSTE24 result in premature aging disorders including Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid diseases. Additional evidence suggests that defects in prelamin A processing, due to diminished ZMPSTE24 expression or activity, may also drive normal physiological aging. Because of the important connection between prelamin A processing and human aging, there is increasing interest in how ZMPSTE24 specifically recognizes and cleaves its substrate prelamin A, encoded by LMNA. Here, we describe two humanized yeast systems we have recently developed to examine ZMPSTE24 processing of prelamin A. These systems differ from one another slightly. Version 1.0 is optimized to analyze ZMPSTE24 mutations, including disease alleles that may affect the function or stability of the protease. Using this system, we previously showed that some ZMPSTE24 disease alleles that affect stability can be rescued by the proteasome inhibitor bortezomib, which may have therapeutic implications. Version 2.0 is designed to analyze LMNA mutations at or near the ZMPSTE24 processing site to assess whether they permit or impede prelamin A processing. Together these systems offer powerful methodology to study ZMPSTE24 disease alleles and to dissect the specific residues and features of the lamin A tail that are required for recognition and cleavage by the ZMPSTE24 protease.

The understudied NIS Synthetase family have been increasingly associated with virulence in bacterial species, due to their key role in the synthesis of hydroxamate and carboxylate "stealth" siderophores. We have identified a model family member, DesD from Streptomyces coelicolor, to characterize structurally using a combination of wild-type and Arg306Gln variant in apo, cofactor product AMP-bound, and cofactor reactant ATP-bound complexes. Kinetics in the family have been limited by solubility and reporter assays, so we have developed a label-free kinetics assay utilizing a single-injection isothermal titration calorimetry-based method. We report second order rate constants 50fold higher than the previous estimations for DesD. Our Arg306Gln DesD variant was also tested in identical buffer and substrate conditions, and confirmed undetectable activity. These are the first reported structures for DesD, and they describe the critical cofactor coordination. This is also the first label-free assay to unambiguously determine kinetics for an NIS synthetase. Introduction:Microbial iron compounds are essential to metabolic processes, but while iron in the environment itself is abundant (Fe 3+ ion in particular), it is not readily available to most microorganisms due to limited solubility (10 -18 M at neutral pH.) 1,2 The bacterial strategy to compete for iron involves the synthesis and secretion of high-affinity small-molecule chelators called siderophores. 3 Siderophores are generally 500-1,000 Da in molecular mass, significantly increase Fe 3+ ion's solubility, and may achieve association constants as high as 10 24 M -1 . 1,2 Aerobic bacteria, facilitative anaerobic bacteria, fungi, and some plants commonly produce at least one siderophore. 1 Several hundred variations have been isolated 4 , and sometimes several siderophores are discovered in the same organism--this apparent functional duplication allows for pathogenic bacteria to circumvent host defenses with varying structures. 5 Most siderophores are

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The integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24’s selectivity for prelamin A.