Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4.

Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical rhomboid intramembrane serine proteases and also others that have lost protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, RHBDL4 (rhomboid-like protein 4) is an endoplasmic reticulum (ER)-resident protease that forms a ternary complex with ubiquitinated substrates and p97/VCP (valosin-containing protein), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the pre-T-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. PTP1B (protein-tyrosine phosphatase nonreceptor type 1, also called PTPN1) was also identified as a potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys63-linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.

HIV-1-induced impairment of dendritic cell cross talk with γδ T lymphocytes.

UNLABELLED: The interplay between dendritic cells (DC) and γδ T lymphocytes represents a network of paracrine and cell contact interactions important for an integrated immune response to pathogens. HIV-1 infection dramatically affects the number and functions of both cell populations, and DC/γδ T cell cross talk may represent a target of virus-induced immune escape. We investigated whether HIV-exposed DC could deliver aberrant signals to interacting γδ T cells. Here we report that the interaction of human γδ T lymphocytes with HIV-1-exposed autologous monocyte-derived DC, but not direct exposure to the virus, impairs lymphocyte expansion and gamma interferon (IFN-γ) production in response to phosphoantigens. This effect is independent of virus strain and occurred in 55% of the donors analyzed. The donor-dependent variation observed relies on the responsiveness of DC to HIV-1 and is strictly related to the capacity of the virus to suppress the maturation-induced expression of interleukin 12 (IL-12). In fact, γδ T cell response to phosphoantigens is almost completely recovered when this cytokine is exogenously added to the DC/lymphocyte cocultures. Interestingly, we show that γδ T lymphocytes are recruited by HIV-1-exposed DC through a CCR5-mediated mechanism and exert a CCL4-mediated control on virus dissemination within DC and susceptible CD4(+) T lymphocytes. These results demonstrate an association between HIV-induced DC dysfunction and alterations of γδ T cell responses. The aberrant cross talk between these two cell populations may contribute to the pathogenesis of HIV infection by further reducing the strength of antiviral immune response. IMPORTANCE: This study provides new evidence on the mechanisms exploited by HIV-1 to evade the host immune response. We report that HIV-1 impairs the cross talk between DC and γδ T lymphocytes, by reducing the capacity of DC to promote functional γδ T cell activation. Interestingly, the virus does not per se interfere with γδ T cell activation, thus highlighting the key role of early DC-HIV-1 interaction in this phenomenon. Furthermore, the results obtained unravel the novel role of γδ T cells in controlling HIV-1 dissemination within the DC population as well as virus transfer to susceptible CD4(+) T lymphocytes. The interactions of DC with innate lymphocytes represent a major control mechanism for an integrated immune response to infection. Understanding how HIV-1 harnesses these pathways may provide important insights on the pathogenesis of disease and offer new opportunities for therapeutic interventions.

Polo-like kinase is necessary for flagellum inheritance in Trypanosoma brucei.

Polo-like kinases play an important role in a variety of mitotic events in mammalian cells, ranging from centriole separation and chromosome congression to abscission. To fulfill these roles, Polo-like kinase homologs move to different cellular locations as the cell cycle progresses, starting at the centrosome, progressing to the spindle poles and then the midbody. In the protist parasite Trypanosoma brucei, the single polo-like kinase homolog T. brucei PLK (TbPLK) is essential for cytokinesis and is necessary for the correct duplication of a centrin-containing cytoskeletal structure known as the bilobe. We show that TbPLK has a dynamic localization pattern during the cell cycle. The kinase localizes to the basal body, which nucleates the flagellum, and then successively localizes to a series of cytoskeletal structures that regulate the position and attachment of the flagellum to the cell body. The kinase localizes to each of these structures as they are duplicating. TbPLK associates with a specialized set of microtubules, known as the microtubule quartet, which might transport the kinase during its migration. Depletion of TbPLK causes defects in basal body segregation and blocks the duplication of the regulators that position the flagellum, suggesting that its presence on these structures might be necessary for their proper biogenesis. TbPLK migrates throughout the cell in T. brucei, but the specific locations to which it targets and its functions are geared towards the inheritance of a properly positioned and attached flagellum.

Dynamic microvilli sculpt bristles at nanometric scale.

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.

Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.

Opposite regulatory effects of IFN-ß and IL-3 on C-type lectin receptors, antigen uptake, and phagocytosis in human macrophages.

CLRs are predominantly expressed in macrophages and myeloid DCs, where they play a key role in antigen recognition, scavenging, and host defense against pathogens. To identify novel immunoregulatory cytokines and networks involved in the control of these functions, we analyzed the coordinate effects of IFN-ß and IL-3 on CLR expression, antigen uptake, and phagocytosis in human MDMs and MDDCs. We report that these cytokines exert opposite regulatory effects on the expression of CLRs and endocytic/phagocytic activities of MDMs. Specifically, IFN-ß markedly inhibits the expression of MR and Dectin-1 during MDM differentiation and impairs the capacity of MDM to internalize antigens and phagocytose unopsonized Candida albicans. Conversely, IL-3 up-modulates MR, Dectin-1, and DC-SIGN, thus allowing more efficient uptake/phagocytosis. Interestingly, IL-3 counteracts the IFN-ß effect on antigen uptake/processing by fully restoring MR expression in IFN-ß-primed MDMs. In contrast, the phagocytic activity is only partially restored as a result of the failure of IL-3 in counteracting IFN-ß-induced Dectin-1 suppression. Notably, IFN-ß-mediated impairment of CLR expression/function occurs in macrophages but not in MDDCs. These results identify IFN-ß and IL-3 as unrecognized regulators of CLR expression and function, unraveling a novel interaction between these cytokines instrumental for the regulation of the macrophage response to pathogens.

An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK in Trypanosoma brucei.

Polo-like kinases are important regulators of cell division, playing diverse roles in mitosis and cytoskeletal inheritance. In the parasite Trypanosoma brucei, the single PLK homologue TbPLK is necessary for the assembly of a series of essential organelles that position and adhere the flagellum to the cell surface. Previous work relied on RNA interference or inhibitors of undefined specificity to inhibit TbPLK, both of which have significant experimental limitations. Here we use an analogue-sensitive approach to selectively and acutely inhibit TbPLK. T. brucei cells expressing only analogue-sensitive TbPLK (TbPLK(as)) grow normally, but upon treatment with inhibitor develop defects in flagellar attachment and cytokinesis. TbPLK cannot migrate effectively when inhibited and remains trapped in the posterior of the cell throughout the cell cycle. Using synchronized cells, we show that active TbPLK is a direct requirement for the assembly and extension of the flagellum attachment zone, which adheres the flagellum to the cell surface, and for the rotation of the duplicated basal bodies, which positions the new flagellum so that it can extend without impinging on the old flagellum. This approach should be applicable to the many kinases found in the T. brucei genome that lack an ascribed function.