Newborn Screen for X-Linked Adrenoleukodystrophy Using Flow Injection Tandem Mass Spectrometry in Negative Ion Mode.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder caused by pathogenic variants in the ATP-binding cassette subfamily D member 1 gene () that encodes the adrenoleukodystrophy protein (ALDP). Defects in ALDP result in elevated cerotic acid, and lead to C26:0-lysophosphatidylcholine (C26:0-LPC) accumulation, which is the primary biomarker used in newborn screening (NBS) for X-ALD. C26:0-LPC levels were measured in dried blood spot (DBS) NBS specimens using a flow injection analysis (FIA) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) performed in negative ion mode.

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2.

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond.

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2.

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond.

TRAF3IP3 negatively regulates cytosolic RNA induced anti-viral signaling by promoting TBK1 K48 ubiquitination.

Innate immunity to nucleic acids forms the backbone for anti-viral immunity and several inflammatory diseases. Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type I interferon (IFN-I). TRAF3-interacting protein 3 (TRAF3IP3, T3JAM) is essential for T and B cell development.

Cotranslational stabilization of Sec62/63 within the ER Sec61 translocon is controlled by distinct substrate-driven translocation events.

The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αβγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation.

Cotranslational folding inhibits translocation from within the ribosome-Sec61 translocon complex.

Eukaryotic secretory proteins cross the endoplasmic reticulum (ER) membrane through a protein-conducting channel contained within the ribosome-Sec61translocon complex (RTC). Using a zinc-finger sequence as a folding switch, we show that cotranslational folding of a secretory passenger inhibits translocation in canine ER microsomes and in human cells. Folding occurs within a cytosolically inaccessible environment, after ER targeting but before initiation of translocation, and it is most effective when the folded domain is 15-54 residues beyond the signal sequence.

The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB.

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3.

Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex.

In eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility, and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA.

Cutting edge: NLRC5-dependent activation of the inflammasome.

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function.

Control of translocation through the Sec61 translocon by nascent polypeptide structure within the ribosome.

During polytopic protein biogenesis, multiple transmembrane segments (TMs) must pass through the ribosome exit tunnel and into the Sec61 translocon prior to insertion into the endoplasmic reticulum membrane. To investigate how movement of a newly synthesized TM along this integration pathway might be influenced by synthesis of a second TM, we used photocross-linking probes to detect the proximity of ribosome-bound nascent polypeptides to Sec61alpha. Probes were inserted at sequential sites within TM2 of the aquaporin-1 water channel by in vitro translation of truncated mRNAs.

Single MHC mutation eliminates enthalpy associated with T cell receptor binding.

The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition.

Global functional analysis of nucleophosmin in Taxol response, cancer, chromatin regulation, and ribosomal DNA transcription.

Analysis of lung cancer response to chemotherapeutic agents showed the accumulation of a Taxol-induced protein that reacted with an anti-phospho-MEK1/2 antibody. Mass spectroscopy identified the protein as nucleophosmin/B23 (NPM), a multifunctional protein with diverse roles: ribosome biosynthesis, p53 regulation, nuclear-cytoplasmic shuttling, and centrosome duplication. Our work demonstrates that following cellular exposure to mitosis-arresting agents, NPM is phosphorylated and its chromatographic property is altered, suggesting changes in function during mitosis.

DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2.

DJ-1/PARK7, a cancer- and Parkinson's disease (PD)-associated protein, protects cells from toxic stresses. However, the functional basis of this protection has remained elusive. We found that loss of DJ-1 leads to deficits in NQO1 [NAD(P)H quinone oxidoreductase 1], a detoxification enzyme.

Criteria for effective design, construction, and gene knockdown by shRNA vectors.

Background: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.

CATERPILLER 16.2 (CLR16.2), a novel NBD/LRR family member that negatively regulates T cell function.

The newly discovered mammalian CATERPILLER (NOD, NALP, PAN) family of proteins share similarities with the NBD-LRR superfamily of plant disease resistance (R) proteins and are predicted to mediate important immune regulatory function. This report describes the first cloning and characterization of a novel CATERPILLER gene, CLR16.2 that is located on human chromosome 16.

Enhancement of CIITA transcriptional function by ubiquitin.

Although increasing evidence indicates that there is a direct link between ubiquitination and mono-ubiquitination and transcription in yeast, this link has not been demonstrated in higher eukaryotes. Here we show that the major histocompatibility complex (MHC) class II transactivator (CIITA), which is required for expression of genes encoding MHC class II molecules, is ubiquitinated. This ubiquitination enhanced the association of CIITA with both MHC class II transcription factors and the MHC class II promoter, resulting in an increase in transactivation function and in the expression of MHC class II mRNA.

Leucine-rich repeats of the class II transactivator control its rate of nuclear accumulation.

Activation of class II major histocompatibility complex (MHC) gene expression is regulated by a master regulator, class II transcriptional activator (CIITA). Transactivation by CIITA requires its nuclear import. This study will address a mechanistic role for the leucine-rich repeats (LRR) of CIITA in regulating nuclear translocation by mutating 12 individual consensus-motif "leucine" residues in both its alpha-motifs and beta-motifs.