SAMHD1's protein expression profile in humans.

The deoxynucleoside triphosphate triphosphohydrolase and 3' → 5' exonuclease SAMHD1 restricts HIV-1 infection in noncycling hematopoietic cells in vitro, and SAMHD1 mutations are associated with AGS. Little is known about the in vivo expression and functional regulation of this cellular factor. Here, we first assessed the SAMHD1 protein expression profile on a microarray of 25 human tissues from >210 donors and in purified primary cell populations. In vivo, SAMHD1 was expressed in the majority of nucleated cells of hematopoietic origin, including tissue-resident macrophages, DCs, pDCs, all developmental stages of thymic T cells, monocytes, NK cells, as well as at lower levels in B cells. Of note, SAMHD1 was abundantly expressed in HIV target cells residing in the anogenital mucosa, providing a basis for its evaluation as a cellular factor that may impact the efficiency of HIV transmission. Next, we examined the effect of the activation status and proinflammatory cytokine treatment of cells on expression and phosphorylation of SAMHD1. Activated, HIV-susceptible CD4(+) T cells carried pSAMHD1(T592), whereas resting CD4(+) T cells and macrophages expressed the unphosphorylated protein with HIV-restrictive activity. Surprisingly, stimulation of these primary cells with IFN-α, IFN-γ, IL-4, IL-6, IL-12, IL-18, IL-27, or TNF-α affected neither SAMHD1 expression levels nor threonine 592 phosphorylation. Only IL-1ß moderately down-regulated SAMHD1 in activated CD4(+) T cells. Taken together, this study establishes the first cross-sectional protein expression profile of SAMHD1 in human tissues and provides insight into its cell cycle-dependent phosphorylation and unresponsiveness to multiple proinflammatory cytokines.

SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells.

Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.

MUF1/leucine-rich repeat containing 41 (LRRC41), a substrate of RhoBTB-dependent cullin 3 ubiquitin ligase complexes, is a predominantly nuclear dimeric protein.

RhoBTB (BTB stands for broad-complex, tramtrack, bric à brac) proteins are tumor suppressors involved in the formation of cullin 3 (Cul3)-dependent ubiquitin ligase complexes. However, no substrates of RhoBTB-Cul3 ubiquitin ligase complexes have been identified. We identified MUF1 (LRRC41, leucine-rich repeat containing 41) as a potential interaction partner of RhoBTB3 in a two-hybrid screening on a mouse brain cDNA library. MUF1 is a largely uncharacterized protein containing a leucine-rich repeat and, interestingly, a BC-box that serves as a linker in multicomponent, cullin 5 (Cul5)-based ubiquitin ligases. We confirmed the interaction of MUF1 with all three mammalian RhoBTB proteins using immunoprecipitation. We characterized MUF1 in terms of expression profile and subcellular localization, the latter also with respect to RhoBTB proteins. We found out that MUF1 is a ubiquitously expressed nuclear protein that, upon coexpression with RhoBTB, partially retains in the cytoplasm, where both proteins colocalize. We also show that MUF1 is able to dimerize similarly to other leucine-rich repeat-containing proteins. To explore the significance of MUF1-RhoBTB interaction within Cul-ligase complexes and the mechanism of MUF1 degradation, we performed a protein stability assay and found that MUF1 is degraded in the proteasome in a Cul5-independent manner by RhoBTB3-Cul3 ubiquitin ligase complex. Finally, we explored a possible heterodimerization of Cul3 and Cul5 and indeed discovered that these two cullins are capable of forming heterodimers. Thus, we have identified MUF1 as the first substrate for RhoBTB-Cul3 ubiquitin ligase complexes. Identification of substrates of these complexes will result in better understanding of the tumor suppressor function of RhoBTB.

Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes--evidence for an autoregulatory mechanism.

RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.

Rho GTPases of the RhoBTB subfamily and tumorigenesis.

RhoBTB proteins constitute a subfamily of atypical members within the Rho family of small guanosine triphosphatases (GTPases). Their most salient feature is their domain architecture: a GTPase domain (in most cases, non-functional) is followed by a prolinerich region, a tandem of 2 broadcomplex, tramtrack, bric a brac (BTB) domains, and a conserved Cterminal region. In humans, the RhoBTB subfamily consists of 3 isoforms: RhoBTB1, RhoBTB2, and RhoBTB3. Orthologs are present in several other eukaryotes, such as Drosophila and Dictyostelium, but have been lost in plants and fungi. Interest in RhoBTB arose when RHOBTB2 was identified as the gene homozygously deleted in breast cancer samples and was proposed as a candidate tumor suppressor gene, a property that has been extended to RHOBTB1. The functions of RhoBTB proteins have not been defined yet, but may be related to the roles of BTB domains in the recruitment of cullin3, a component of a family of ubiquitin ligases. A model emerges in which RhoBTB proteins are required to maintain constant levels of putative substrates involved in cell cycle regulation or vesicle transport through targeting for degradation in the 26S proteasome. RhoBTB proteins are engrossing the list of Rho GTPases involved in tumorigenesis. Unlike typical Rho GTPases (usually overexpressed or hyperactive), RhoBTB proteins appear to play a part in the carcinogenic process through a mechanism that involves the decreased or abolished expression of the corresponding genes, or more rarely, mutations that result in impaired functioning of the protein, presumably leading to the accumulation of RhoBTB substrates and alterations of the cellular homeostasis.