Biomedical subjects

Karla E Marz

Publications and source records attributed to Karla E Marz.

5 recordsLinked to original sources

PubMed2007

Structure/function analysis of Xenopus cryptochromes 1 and 2 reveals differential nuclear localization mechanisms and functional domains important for interaction with and repression of CLOCK-BMAL1.

Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.

ARNTL Transcription Factors↗

PubMed2006

SNARE complex zero layer residues are not critical for N-ethylmaleimide-sensitive factor-mediated disassembly.

Membrane-anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNARE complexes are parallel four-helix bundles stabilized in part by hydrophobic interactions within their core. At the center of SNARE complexes is a distinctive zero layer that consists of one arginine and three glutamines. This zero layer is thought to play a special role in the biology of the SNARE complex. One proposal is that the polar residues of the zero layer enable N-ethylmaleimide-sensitive factor (NSF)-mediated SNARE complex disassembly. Here, we studied the effects of manipulating the zero layer of the well studied synaptic SNARE complex in vitro and in vivo. Using a fluorescence-based assay to follow SNARE complex disassembly in real time, we found that the maximal rate at which NSF disassembles complexes was unaffected by mutations in the zero layer, including single replacement of the syntaxin glutamine with arginine as well as multiple replacement of all four layer residues with non-polar amino acids. To determine whether syntaxin with arginine instead of glutamine in its zero layer can support SNARE function in vivo, we introduced it as a transgene into a Caenorhabditis elegans syntaxin-null strain. Mutant syntaxin rescued viability and locomotory defects similarly to wild-type syntaxin, demonstrating that SNARE complexes with two glutamines and two arginines in the zero layer can support neurotransmission. These findings show that residues of the zero layer do not play an essential role in NSF-mediated disassembly.

Aldicarb↗

PubMed2004

The hyh mutation uncovers roles for alpha Snap in apical protein localization and control of neural cell fate.

The hyh (hydrocephalus with hop gait) mouse shows a markedly small cerebral cortex at birth and dies postnatally from progressive enlargement of the ventricular system. Here we show that the small hyh cortex reflects altered cell fate. Neural progenitor cells withdraw prematurely from the cell cycle, producing more early-born, deep-layer cerebral cortical neurons but depleting the cortical progenitor pool, such that late-born, upper-layer cortical neurons are underproduced, creating a small cortex. hyh mice carry a hypomorphic missense mutation in the gene Napa encoding soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein alpha (alpha Snap), involved in SNAP receptor (SNARE)-mediated vesicle fusion in many cellular contexts. A targeted null Napa mutation is embryonically lethal. Altered neural cell fate is accompanied by abnormal localization of many apical proteins implicated in regulation of neural cell fate, including E-cadherin, beta-catenin, atypical protein kinase C (aPKC) and INADL (inactivation-no-afterpotential D-like, also known as protein associated with Lin7, or Pals1). Apical localization of the SNARE Vamp7 is also disrupted. Thus, alpha Snap is essential for apical protein localization and cell fate determination in neuroepithelial cells.

Animals↗

PubMed2003

Defining the SNARE complex binding surface of alpha-SNAP: implications for SNARE complex disassembly.

N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein alpha-soluble NSF attachment protein (alpha-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of alpha-SNAP in this process, we used site-directed mutagenesis to identify residues in alpha-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine alpha-helices of alpha-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced alpha-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between alpha-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and alpha-SNAP work together to drive disassembly.

Amino Acid Sequence↗

PubMed2002

Sealed with a twist: complexin and the synaptic SNARE complex.

Knockout experiments show that complexins are crucial for Ca(2+)-dependent neurotransmitter release. In the recently solved crystal structure of complexin I bound to the synaptic SNARE complex, complexin binds along the groove between the syntaxin and synaptobrevin coils. This structure, together with NMR data, suggests that the role of complexin is to stabilize the SNARE complex as it forms from SNAREs in vesicle and target membranes.

Adaptor Proteins, Vesicular Transport↗