HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization.

Abnormal promoter DNA methylation in juvenile myelomonocytic leukemia is not caused by mutation in DNMT3ASeveral recent publications reported the occurrence of mutations in the DNA methyltransferase 3A (DNMT3A) gene in acute myeloid leukemia (AML). [1][2][3] The majority of DNMT3A mutations identified affect residue R882 which is located within the methyltransferase domain. 1,2 A strong predilection for leukemias of monocytic or myelomonocytic lineage was observed. 2,3 This, and our previous findings linking altered DNA methylation patterns with poor prognosis in juvenile myelomonocytic leukemia (JMML), 4 led us to hypothesize that somatic DNMT3A mutations might also occur in JMML.We bidirectionally sequenced DNMT3A exon 23 (containing the hotspot codon 882) in granulocyte DNA from 113 JMML patients. All children were enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) studies 98 or 2006, and informed consent had been obtained from patients' guardians. The patients' median age was 2.0 years. The hotspot mutation DNMT3A p.R882H was identified in JMML patient D101 (age at diagnosis 6.5 years, somatic NRAS G12V mutation; Figure 1A), while all other samples exhibited wild-type sequence. Epstein-Barr virus-transformed B lymphocytes of D101 carried wild-type sequence, indicating somatic origin of the DNMT3A mutation.Although DNMT3A is generally overexpressed in AML blasts versus healthy leukocytes, 5 the expression does not correlate with occurrence of DNMT3A mutations. 1 Despite the finding that DNMT3A codon 882 mutations lead to reduced enzymatic activity, 2 the genome-wide DNA methylation pattern of DNMT3A codon 882-mutant AML cells does not differ significantly from that of DNMT3A wild-type blasts. 1 We measured DNMT3A mRNA expression in mononuclear cells from 9 JMML patients by quantitative real-time PCR (RNA from patient D101 was unavailable). The abundance of the DNMT3A transcript in JMML cells varied considerably, with some cases expressing DNMT3A at higher levels than leukocytes from healthy subjects, and others at lower levels (range, 0.08-14.05 fold; Figure 1B). Unlike in AML where uniform blast populations are studied, our results may reflect the heterogeneous cellular composition of JMML samples. DNA methylation data were available for 6 of the 9 JMML samples studied, 4 but there was no association of high DNMT3A expression with DNA hypermethylation ( Figure 1C). Remarkably, leukemic cells of D101 carried hypermethylated BMP4, CALCA and CDKN2B promoters 4 despite the presence of a DNMT3A loss-of-function mutation.Several investigators highlighted the possible pathophysiologic role of the DNA-methylating enzyme DNMT3A in myeloid malignant disorders other than JMML. [1][2][3]6,7 We conclude that genetic or transcriptional aberrations of DNMT3A do not contribute to leukemogenesis in JMML. While the recurrent nature of DNMT3A alterations in AML, the strong preference to target codon 882 and the prognostic significance argue in favor of non-random pathogenetic relevance in AML, a DNA methylat...

Protein-ligand interactions characterise and govern the current state and fate of a living cell. The specificity of proteins is mainly determined by the relative affinities to each potential ligand. To investigate the consequences and potentials of ligands with increased specificity in comparison with ligands optimised solely for affinity, it was necessary to identify ligands that are optimised towards specificity instead of a barely optimised affinity to a given target. In the presented example, a modified phage display screening procedure yielded specific ligands for the LckSH3 domain. We found that increased specificity of one of the hereby obtained ligands for LckSH3 is achieved at the cost of a slightly reduced affinity to LckSH3 and a drastically reduced affinity to other SH3 domains. A surface plasmon resonance experiment simulating in vivo-like realistic competitive binding conditions exerted enhanced binding behaviour of the specific ligand under these binding conditions. The experimental data, together with a mathematical model describing the complex experimental situation, and theoretical considerations lead to the conclusion that increased specificity is achieved at the cost of reduced affinity, but after all, it pays if the ligand is applied under realistic, i.e. competitive, conditions.