Journal of Biological Chemistry

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase (PDI) family member that accelerates ER-associated degradation (ERAD) by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates...

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia...

Imbalanced immune responses are a prominent hallmark of cancer and autoimmunity. Overactive as well as suppressed immunological activity is evidenced to promote disease. This immunological disbalance is often driven through disrupted immunomodulation by innate myeloid immune cells. Myeloid cells can be overly suppressive, inhibiting protective immune responses or be inactive not controlling autoreactive immune cells. Understanding the mechanisms that induce and drive suppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (TolDCs), can facilitate the development of immune-restoring therapeutic approaches in patients...

The double nucleation mechanism of amyloid β (Aβ) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF), but with reduced rate of all microscopic processes. Here we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aβ42 fibril formation as a function of time in the absence and presence of ApoA-I, recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles...

The cytoplasmic accumulation of the nuclear protein TDP-43 has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration (FTLD). TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of FTLD...

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus (RSV)-approved monoclonal antibody Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH ) as synthetic receptors...

Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain non-genomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes (T1D). A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets...

Mosaicism refers to the presence of genetically distinct cell populations in an individual derived from a single zygote, which occurs during the process of development, aging, and genetic diseases. To date, a variety of genetically engineered mosaic analysis models have been established and widely used in studying gene function at exceptional cellular and spatio-temporal resolution, leading to many ground-breaking discoveries. Mosaic analysis with a repressible cellular marker (MARCM) and mosaic analysis with double markers (MADM) are genetic mosaic analysis models based on trans-recombination...

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli...

Herbicides are small molecules that act by inhibiting specific molecular target sites within primary plant metabolic pathways resulting in catastrophic and lethal consequences. The stress induced by herbicides generates reactive oxygen species (ROS), but little is known about the nexus between each herbicide mode of action (MoA) and their respective ability to induce ROS formation. Indeed, some herbicides cause dramatic surges in ROS levels as part of their primary MoA, whereas other herbicides may generate some ROS as a secondary effect of the stress they imposed on plants...

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system...

Vanilloids such as capsaicin and resiniferatoxin (RTX) are highly selective and potent activators for TRPV1, a nociceptor for heat and pain perception. However, the intrinsic vanilloid binding affinity, key for understanding TRPV1 function, remains unknown despite intensive investigations by electrophysiological, structural, and computational methods. In this study, we determined capsaicin binding affinity under physiological conditions by isolating individual binding steps with concatemers. We estimated the capsaicin association constant of a wildtype subunit to be in the order of 106 M-1 , and that of the Y511A mutant subunit to be a hundred times lower, in the order of 104 M-1 ...

Over 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semi-crystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies...

The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome...

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins...

The kinesin-14 motor proteins play important roles in tumor development and drug resistance and have been reported as potential biomarkers or therapeutic targets for tumor treatment. However, KIFC2, one of the kinesin-14 motor family members, remains largely unknown in prostate cancer (PCa) progression. Here, we used the GEO and TCGA datasets, western blotting, and IHC analyses were used to detect KIFC2 expression in PCa tissues. Additionally, a series of in vivo and in vitro experiments were utilized to demonstrate the roles of KIFC2 in PCa cells...

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose and UDP-GalNAc. Structural comparison with the homologue from Escherichia coli (EcWaaG) revealed five key differences in the sugar binding pocket. Solution-state NMR analysis showed that wildtype PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose...

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared to the currently used method...

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VH Hs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VH Hs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis...

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