Accuracy in mineral identification: image spectral and spatial resolutions and mineral spectral properties

Problems related to airborne hyperspectral image data are reviewed and the requirements for data analysis applied to mineralogical (rocks and soils) interpretation are discussed. The variability of mineral spectral features, including absorption position, shape and depth is considered and interpreted as due to chemical composition, grain size effects and mineral association. It is also shown how this variability can be related to well defined geologic processes. The influence of sensor noise and diffuse atmospheric radiance in classification accuracy is also analyzed

Zinc is essential for high-affinity DNA binding and recombinase activity of φC31 integrase

The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of ϕC31 integrase (Int) and its relatives indicate the presence of a conserved motif containing four cysteines resembling a zinc finger. Inductively coupled plasma–mass spectrometry (ICP–MS) confirmed that an Int monomer contains one atom of zinc. Pre-incubation of Int with ethylenediaminetetraacetic acid (EDTA) was detrimental for both recombination activity and DNA binding affinities but full activity could be restored by adding back Zn2+. Mutations in the cysteines and other highly conserved residues yielded proteins that were hypersensitive to proteases, suggesting that without zinc the domain is unfolded. Substitutions in the highly charged region between the conserved cysteines led to lowered DNA binding affinities while circular dichroism revealed that these variant Ints were not greatly affected in overall folding. Int was protected from inhibition by EDTA when DNA containing an attachment site was present suggesting that the zinc finger and the DNA are in close proximity. A truncated mutant of Int, hInt V371SUGA, lacking the putative zinc finger could bind DNA with low affinity. The data are consistent with there being at least two DNA binding motifs in Int one of which is the zinc finger-like motif

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.Publisher PDFPeer reviewe

Crystallographic and Biochemical Analysis of the Ran-Binding Zinc Finger Domain

The nuclear pore complex (NPC) resides in circular openings within the nuclear envelope and serves as the sole conduit to facilitate nucleocytoplasmic transport in eukaryotes. The asymmetric distribution of the small G protein Ran across the nuclear envelope regulates directionality of protein transport. Ran interacts with the NPC of metazoa via two asymmetrically localized components, Nup153 at the nuclear face and Nup358 at the cytoplasmic face. Both nucleoporins contain a stretch of distinct, Ran-binding zinc finger domains. Here, we present six crystal structures of Nup153-zinc fingers in complex with Ran and a 1.48 Å crystal structure of RanGDP. Crystal engineering allowed us to obtain well diffracting crystals so that all ZnF–Ran complex structures are refined to high resolution. Each of the four zinc finger modules of Nup153 binds one Ran molecule in apparently non-allosteric fashion. The affinity is measurably higher for RanGDP than for RanGTP and varies modestly between the individual zinc fingers. By microcalorimetric and mutational analysis, we determined that one specific hydrogen bond accounts for most of the differences in the binding affinity of individual zinc fingers. Genomic analysis reveals that only in animals do NPCs contain Ran-binding zinc fingers. We speculate that these organisms evolved a mechanism to maintain a high local concentration of Ran at the vicinity of the NPC, using this zinc finger domain as a sink

A tool for examining the role of the zinc finger myelin transcription factor 1 (Myt1) in neural development: Myt1 knock-in mice

The Myt1 family of transcription factors is unique among the many classes of zinc finger proteins in how the zinc-stabilized fingers contact the DNA helix. To examine the function of Myt1 in the developing nervous system, we generated mice in which Myt1 expression was replaced by an enhanced Green Fluorescent Protein fused to a Codon-improved Cre recombinase as a protein reporter. Myt1 knock-in mice die at birth, apparently due to improper innervation of their lungs. Elimination of Myt1 did not significantly affect the number or distribution of neural precursor cells that normally express Myt1 in the embryonic spinal cord. Nor was the general pattern of differentiated neurons altered in the embryonic spinal cord. The Myt1 knock-in mice should provide an important tool for identifying the in vivo targets of Myt1 action and unraveling the role of this structurally distinct zinc finger protein in neural development

Minimal Functional Sites Allow a Classification of Zinc Sites in Proteins

Zinc is indispensable to all forms of life as it is an essential component of many different proteins involved in a wide range of biological processes. Not differently from other metals, zinc in proteins can play different roles that depend on the features of the metal-binding site. In this work, we describe zinc sites in proteins with known structure by means of three-dimensional templates that can be automatically extracted from PDB files and consist of the protein structure around the metal, including the zinc ligands and the residues in close spatial proximity to the ligands. This definition is devised to intrinsically capture the features of the local protein environment that can affect metal function, and corresponds to what we call a minimal functional site (MFS). We used MFSs to classify all zinc sites whose structures are available in the PDB and combined this classification with functional annotation as available in the literature. We classified 77% of zinc sites into ten clusters, each grouping zinc sites with structures that are highly similar, and an additional 16% into seven pseudo-clusters, each grouping zinc sites with structures that are only broadly similar. Sites where zinc plays a structural role are predominant in eight clusters and in two pseudo-clusters, while sites where zinc plays a catalytic role are predominant in two clusters and in five pseudo-clusters. We also analyzed the amino acid composition of the coordination sphere of zinc as a function of its role in the protein, highlighting trends and exceptions. In a period when the number of known zinc proteins is expected to grow further with the increasing awareness of the cellular mechanisms of zinc homeostasis, this classification represents a valuable basis for structure-function studies of zinc proteins, with broad applications in biochemistry, molecular pharmacology and de novo protein design

MYT1L mutations cause intellectual disability and variable obesity by dysregulating gene expression and development of the neuroendocrine hypothalamus

Deletions at chromosome 2p25.3 are associated with a syndrome consisting of intellectual disability and obesity. The smallest region of overlap for deletions at 2p25.3 contains PXDN and MYT1L. MYT1L is expressed only within the brain in humans. We hypothesized that single nucleotide variants (SNVs) in MYT1L would cause a phenotype resembling deletion at 2p25.3. To examine this we sought MYT1L SNVs in exome sequencing data from 4, 296 parent-child trios. Further variants were identified through a genematcher-facilitated collaboration. We report 9 patients with MYT1L SNVs (4 loss of function and 5 missense). The phenotype of SNV carriers overlapped with that of 2p25.3 deletion carriers. To identify the transcriptomic consequences of MYT1L loss of function we used CRISPR-Cas9 to create a knockout cell line. Gene Ontology analysis in knockout cells demonstrated altered expression of genes that regulate gene expression and that are localized to the nucleus. These differentially expressed genes were enriched for OMIM disease ontology terms “mental retardation”. To study the developmental effects of MYT1L loss of function we created a zebrafish knockdown using morpholinos. Knockdown zebrafish manifested loss of oxytocin expression in the preoptic neuroendocrine area. This study demonstrates that MYT1L variants are associated with syndromic obesity in humans. The mechanism is related to dysregulated expression of neurodevelopmental genes and altered development of the neuroendocrine hypothalamus

Dual Functions of ASCIZ in the DNA Base Damage Response and Pulmonary Organogenesis

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis

Domain Analysis Reveals That a Deubiquitinating Enzyme USP13 Performs Non-Activating Catalysis for Lys63-Linked Polyubiquitin

Deubiquitination is a reverse process of cellular ubiquitination important for many biological events. Ubiquitin (Ub)-specific protease 13 (USP13) is an ortholog of USP5 implicated in catalyzing hydrolysis of various Ub chains, but its enzymatic properties and catalytic regulation remain to be explored. Here we report studies of the roles of the Ub-binding domains of USP13 in regulatory catalysis by biochemical and NMR structural approaches. Our data demonstrate that USP13, distinct from USP5, exhibits a weak deubiquitinating activity preferring to Lys63-linked polyubiquitin (K63-polyUb) in a non-activation manner. The zinc finger (ZnF) domain of USP13 shares a similar fold with that of USP5, but it cannot bind with Ub, so that USP13 has lost its ability to be activated by free Ub. Substitution of the ZnF domain with that of USP5 confers USP13 the property of catalytic activation. The tandem Ub-associated (UBA) domains of USP13 can bind with different types of diUb but preferentially with K63-linked, providing a possible explanation for the weak activity preferring to K63-polyUb. USP13 can also regulate the protein level of CD3δ in cells, probably depending on its weak deubiquitinating activity and the Ub-binding properties of the UBA domains. Thus, the non-activating catalysis of USP13 for K63-polyUb chains implies that it may function differently from USP5 in cellular deubiquitination processes