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Background: This paper describes an automated method for finding clusters of interconnected proteins in protein interaction networks and retrieving protein annotations associated with these clusters. Results: Protein interaction graphs were separated into subgraphs of interconnected proteins, using the JUNG implementation of Girvan and Newman's Edge-Betweenness algorithm. Functions were sought for these subgraphs by detecting significant correlations with the distribution of Gene Ontology terms which had been used to annotate the proteins within each cluster. The method was implemented using freely available software (JUNG and the R statistical package). Protein clusters with significant correlations to functional annotations could be identified and included groups of proteins know to cooperate in cell metabolism. The method appears to be resilient against the presence of false positive interactions. Conclusion: This method provides a useful tool for rapid screening of small to medium size protein interaction datasets. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed. [ABSTRACT FROM AUTHOR]

The availability of comprehensive protein-protein interaction maps will significantly enhance medical research and aid the functional characterisation of novel genes. To date, the largest scale studies of protein-protein interactions have used the yeast two hybrid method. In this review we take a closer look at the different approaches used in these studies and discuss some key considerations that should be taken into account when designing high throughput interaction mapping projects. [ABSTRACT FROM PUBLISHER]

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture. [ABSTRACT FROM AUTHOR]

High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products. HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed. We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases. The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1. Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome. Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality. [Copyright &y& Elsevier]

Recent studies revealed that CD39 was highly expressed in tumor-specific CD4 + tumor infiltrating lymphocytes (TILs). However, the divergent function of CD39 + T cells remains to be elucidated in colorectal cancer (CRC). In this study, T cells from CRC patients and tumor-bearing mice were isolated to evaluate the function of CD39 in T cells. We found that CD39 was elevated in intratumoral T cells from CRC patients, and negatively correlated with cytokine secretion capacity. T cell activation induced CD39 expression, and CD39 + T cells produced more IFN-γ in response to CRC tumor antigens. In addition, CD39 + T cells in the spleens of tumor-bearing mice exhibited a stronger anti-tumor activity in vitro than CD39 - T cells, but there was no significant difference in the anti-tumor activities between CD39 - TILs and CD39 + TILs. Moreover, we found that CD39 + T cells expressed higher checkpoint molecules and contained a higher proportion of Treg cells than CD39 - T cells, suggesting that CD39 + T cells may be correlated with an immunosuppressive phenotype. And CD39 expression on T cells could convert pro-inflammatory eATP to immunosuppressive eADO. However, both T cells from the vaccinated-wild-type mice and CD39 -/- mice could recognize and eliminate tumor cells in vitro, and adoptive transfer of these T cells resulted in tumor growth inhibition in tumor-bearing mice. In conclusion, our study revealed the divergent functions of CD39 + T cells, which were reactive to tumor antigen but exhibited a dysfunctional phenotype., Competing Interests: None., (AJCR Copyright © 2024.)

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed.

The inhibitory kappaB (IkappaB)-like (IkappaBL) gene is located within the Class III region of the MHC on human chromosome 6. Previous analysis of the predicted amino acid sequence of the human IkappaBL protein revealed three putative functional domains; 2-3 ankyrin repeat sequences, which are similar to the second and third ankyrin repeats of the nuclear factor kappaB (NF-kappaB) protein; three PEST sequence motifs (a sequence that is rich in proline, serine, aspartic acid and threonine residues), which are also found in other IkappaB family members; and a C-terminal leucine zipper-like motif. In the present study we have identified a novel bipartite motif, which is required for nuclear localization of the IkappaBL protein. Analyses of IkappaBL-specific transcripts revealed the existence of a widely expressed spliced variant form of IkappaBL (IkappaBLsv1), which lacks the amino acid sequence GELEDEWQEVMGRFE (where single-letter amino-acid notation has been used). Interestingly, translation of IkappaBL mRNA in vivo was found to initiate predominantly from the second available methionine, thereby resulting in the disruption of the predicted N-terminal PEST sequence. Also, transient expression of T7 epitope-tagged IkappaBL and IkappaBLsv1 proteins in mammalian cells showed that both proteins were targeted to the nucleus, where they accumulate in nuclear speckles. To define the protein domains required for nuclear import and subnuclear localization, a complementary set of deletion mutants and enhanced green fluorescent protein-IkappaBL domain fusions were expressed in mammalian cells. Data from these experiments show that a combination of the ankyrin-repeat region and an adjacent arginine-rich sequence are necessary and sufficient for both nuclear import and speckle localization.