GTPrS-dependent phospholipase D activity in human neutrophils was investigated using exogenous phospholipid as a substrate. Both cytosolic and membrane- associated phospholipase D activities were identified. The previously described 50 kDa cytosolic activating factor was resolved chromatographically from the cytosolic phospholipase D. Using exogenous phospholipid as substrate along with chromatographically resolved 50 kDa factor and recombinant ADP-ribosylation factor 1, plasma membrane was required for activity, indicating that the activity which was previously seen using endogenous phospholipid substrate was due to a phospholipase D located in the plasma membrane. In addition, ADP-ribosylation factor and the 50 kDa factor activated synergistically. Using neutrophil plasma membranes, a third regulator of neutrophil membrane phospholipase D was identified from bovine brain cytosol. This factor was resolved from ADP-ribosylation factor and Rho A by successive column chromatographies. The brain factor showed a synergistic effect with the 50 kDa neutrophil activator but an additive effect with recombinant ADP- ribosylation factor. Whether or not ADP-ribosylation factor or the brain factor were present, high activities were seen only when the 50 kDa factor was present, indicating that the 50 kDa cytosolic factor is a major activating factor for the neutrophil plasma membrane phospholipase D.
ADP-Ribosylation Factor 1 ; ADP-Ribosylation Factors* ; Brain* ; Cell Membrane ; Chromatography ; Cytosol* ; Fibrinogen ; Humans ; Membranes ; Neutrophils* ; Phospholipase D* ; Phospholipases*

The morbidity of neurodegenerative diseases are increased in recent years, however, the treatment is limited. Poly ADP-ribosylation (PARylation) is a post-translational modification of protein that catalyzed by poly(ADP-ribose) polymerase (PARP). Studies have shown that PARylation is involved in many neurodegenerative diseases such as stroke, Parkinson's diseases, Alzheimer's disease, amyotrophic lateral sclerosis and so on, by affecting intracellular translocation of protein molecules, protein aggregation, protein activity, and cell death. PARP inhibitors have showed neuroprotective efficacy for neurodegenerative diseases in pre-clinical studies and phase Ⅰ clinical trials. To find new PARP inhibitors with more specific effects and specific pharmacokinetic characteristics will be the new direction for the treatment of neurodegenerative diseases. This paper reviews the recent progress on PARylation in neurodegenerative diseases.
ADP-Ribosylation ; Humans ; Neurodegenerative Diseases ; physiopathology ; Poly Adenosine Diphosphate Ribose ; Poly(ADP-ribose) Polymerases ; metabolism

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
ADP-Ribosylation Factor 1 ; chemistry ; metabolism ; Animals ; Coatomer Protein ; chemistry ; metabolism ; Cytosol ; chemistry ; metabolism ; GTPase-Activating Proteins ; chemistry ; metabolism ; Humans ; Membranes, Artificial ; Rats

Adenosine diphosphate (ADP)-ribosylation is a unique post-translational modification that regulates many biological processes, such as DNA damage repair. During DNA repair, ADP-ribosylation needs to be reversed by ADP-ribosylhydrolases. A group of ADP-ribosylhydrolases have a catalytic domain, namely the macrodomain, which is conserved in evolution from prokaryotes to humans. Not all macrodomains remove ADP-ribosylation. One set of macrodomains loses enzymatic activity and only binds to ADP-ribose (ADPR). Here, we summarize the biological functions of these macrodomains in DNA damage repair and compare the structure of enzymatically active and inactive macrodomains. Moreover, small molecular inhibitors have been developed that target macrodomains to suppress DNA damage repair and tumor growth. Macrodomain proteins are also expressed in pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these domains may not be directly involved in DNA damage repair in the hosts or pathogens. Instead, they play key roles in pathogen replication. Thus, by targeting macrodomains it may be possible to treat pathogen-induced diseases, such as coronavirus disease 2019 (COVID-19).
ADP-Ribosylation ; COVID-19/metabolism* ; DNA Repair/physiology* ; Evolution, Molecular ; Humans ; Models, Biological ; Models, Molecular ; N-Glycosyl Hydrolases/metabolism* ; Poly(ADP-ribose) Polymerases/metabolism* ; Protein Domains ; SARS-CoV-2/pathogenicity*

OBJECTIVETo detect expression of mouse ARL-1 homologous proteins in mouse tissues, and analyze homology, genetic distance and phylogenetic relationship between human aldose reductase like-1 (ARL-1) and mouse homologous proteins.

METHODSHomology of mouse ARL-1 homologous proteins with human ARL-1 was analyzed by software Clustal X 1.8 using GenBank and Swiss-Prot database; genetic distance and phylogenetic relationship between mouse ARL-1 homologous proteins and human ARL-1 were analyzed by software Mega 2.0; mouse tissues were detected by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits.

RESULTSThe amino acid sequence of human ARL-1 was 83%, 82%, 81%, 79%, 70%, 51%, 50% and 45% identical to that of the Chinese hamster ovary reductase (CHO-Red), the mouse fibroblast growth factor-regulated protein (FR-1), rat aldose reductase-like protein (rARLP), the mouse vas deferens protein (MVDP), rat lens aldose reductase (LeAR), delta4-3-ketosteroid-5beta-reductase (5beta-Red), rat aldo-keto reductase protein c (RaK-c) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Among all the mouse ARL-1 homologous proteins, the genetic distance between CHO-Red and human ARL was the shortest (18.0%, P = 0.023), next was FR-1 (19.1%, P=0.023) and rARLP (19.9%, P = 0.025). From the phylogenetic tree, the protein whose relationship with human ARL-1 was the closest with CHO-Red, next was mouse FR-1, rARLP, MVDP and LeAR. Homologous proteins were found in mouse tissues including vas deferens, testis, bladder and uterus by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits.

CONCLUSIONSCHO-Red has the highest homology, the shortest genetic distance and the closest relationship with human ARL-1, next is FR-1, rARLP, MVDP. The major distribution of mouse ARL-1 homologous proteins is in vas deferens, testis, bladder and uterus, deducing they might be CHO-Red, FR-1, rARLP or MVDP

ADP-Ribosylation Factors ; biosynthesis ; genetics ; Aldehyde Reductase ; biosynthesis ; genetics ; Animals ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Female ; Gene Expression ; Humans ; Male ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; genetics ; Pregnancy ; Sequence Homology, Amino Acid

OBJECTIVETo investigate the effects of silencing ADP-ribosylation factor 6 (Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC-3 and the possible molecular mechanisms.

METHODSThree Arf6-specific small interfering RNA (siRNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by real-time PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC-3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting.

RESULTSTransfection of siRNA-3 resulted in significantly decreased Arf6 mRNA and protein expression with inhibition rates of (91.88±3.13)% and (86.37±0.57)%, respectively. Arf6 silencing by siRNA-3 markedly suppressed the proliferation, migration and invasion of PC-3 cells and reduced the expression levels of p-ERK1/2 and Rac1.

CONCLUSIONSilencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC-3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.

ADP-Ribosylation Factors ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Humans ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neoplasm Invasiveness ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; Wound Healing ; rac1 GTP-Binding Protein ; metabolism

As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.
ADP-Ribosylation Factors/metabolism/physiology ; Animals ; Basic Helix-Loop-Helix Transcription Factors/metabolism/physiology ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Glucose/*pharmacology ; Insulin/*secretion ; Insulin-Secreting Cells/*drug effects/enzymology/metabolism/secretion ; Intracellular Signaling Peptides and Proteins/metabolism/physiology ; Mice ; Models, Biological ; Oligodeoxyribonucleotides, Antisense/pharmacology ; Phospholipase D/antagonists & inhibitors/genetics/metabolism/*physiology ; Protein-Serine-Threonine Kinases/metabolism/physiology ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism/physiology ; Signal Transduction/drug effects/genetics