Subunit organization and Rab interactions of Vps-C protein complexes that control endolysosomal membrane traffic

Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their signaling activities. To better understand these large and intricate complexes, we performed interaction surveys to assemble domain-level interaction topologies for the eight Vps-C subunits. We identified numerous intersubunit interactions and up to six Rab-binding sites. Functional modules coordinate the major Rab interactions within CORVET and HOPS. The CORVET-specific subunits, Vps3 and Vps8, form a subcomplex and physically and genetically interact with the Rab5 orthologue Vps21. The HOPS-specific subunits, Vps39 and Vps41, also form a subcomplex. Both subunits bind the Rab7 orthologue Ypt7, but with distinct nucleotide specificities. The in vivo functions of four RING-like domains within Vps-C subunits were analyzed and shown to have distinct functions in endolysosomal transport. Finally, we show that the CORVET- and HOPS-specific subunits Vps3 and Vps39 bind the Vps-C core through a common region within the Vps11 C-terminal domain (CTD). Biochemical and genetic experiments demonstrate the importance of these regions, revealing the Vps11 CTD as a key integrator of Vps-C complex assembly, Rab signaling, and endosomal and lysosomal traffic.     

Microarray and RNA in situ hybridization assay for recurrence risk markers of breast carcinoma and ductal carcinoma in situ: Evidence supporting the use of diverse pathways panels

Breast tumor stratification by recurrence-risk is critical for deciding patient treatment. Here an approach combining cancer pathways microarray data complemented by RNA in situ hybridization (ISH) was investigated as a means for recurrence marker discovery and visualization in pathology specimens. LncRNA and mRNA expressions in breast carcinomas with low (n = 8) vs intermediate/high (n = 10) recurrence-scores as estimated by 21-gene assay and pathology review were compared by microarray assay. Tissue microarrays were prepared from breast carcinomas (n = 20) and ductal carcinoma in situ (DCIS) specimens (n = 84 patients) with known outcomes. Thirteen RNA ISH assays were performed: lncRNAs (BBC3-1, FER3, RAD21-AS1, ZEB1-2) and mRNAs (GLO1, GLTSCR2, TGFB1, TLR2) (implicated by the microarray data); MKI67; a pooled panel of recurrence-associated proliferation markers (BIRC5, Cyclin B1, MKI67, MYBL2, STK15); a pooled panel of non-proliferation recurrence-associated markers (CEACAM5, HTF9C, NDRG1, TP53, SLC7A5); and lncRNAs H19 and HOTAIR. Seven lncRNAs and 10 mRNAs showed significantly (P < .05) altered upregulation or downregulation by microarray assay: carcinoma RNA ISH staining did not mirror these patterns. HOTAIR staining was associated with a higher breast cancer recurrence score (P = .0152); qualitatively, H19 was massively expressed in a metaplastic triple negative breast carcinoma. Among the DCIS cohort, significant associations with multiple outcome variables were noted for TGFB1 and the non-proliferation panel (P-value range: .0001 to .047); proliferation panel staining showed an association with increasing DCIS grade (P = .0269) but not with outcomes. The findings support recurrence-risk estimation by the use of multi-marker panels that are representative of diverse cellular pathways rather than over-reliance on proliferation targets. H19, HOTAIR, and TGFB1 RNA ISH show potential for selective diagnostics.     

Deletion of the monkeypox virus inhibitor of complement enzymes locus impacts the adaptive immune response to monkeypox virus in a nonhuman primate model of infection

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.     

Zebrafish relatively relaxed mutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease

Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca(2+) transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca(2+) release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.