Alkylating HIV-1 Nef - a potential way of HIV intervention
© Jin et al; licensee BioMed Central Ltd. 2010
Received: 1 February 2010
Accepted: 26 July 2010
Published: 26 July 2010
Nef is a 27 KDa HIV-1 accessory protein. It downregulates CD4 from infected cell surface, a mechanism critical for efficient viral replication and pathogenicity. Agents that antagonize the Nef-mediated CD4 downregulation may offer a new class of drug to combat HIV infection and disease. TPCK (N-α-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-α-p-tosyl-L-lysine chloromethyl ketone) are alkylation reagents that chemically modify the side chain of His or Cys residues in a protein. In search of chemicals that inhibit Nef function, we discovered that TPCK and TLCK alkylated HIV Nef.
Nef modification by TPCK was demonstrated on reducing SDS-PAGE. The specific cysteine residues modified were determined by site-directed mutagenesis and mass spectrometry (MS). The effect of TPCK modification on Nef-CD4 interaction was studied using fluorescence titration of a synthetic CD4 tail peptide with recombinant Nef-His protein. The conformational change of Nef-His protein upon TPCK-modification was monitored using CD spectrometry
Incubation of Nef-transfected T cells, or recombinant Nef-His protein, with TPCK resulted in mobility shift of Nef on SDS-PAGE. Mutagenesis analysis indicated that the modification occurred at Cys55 and Cys206 in Nef. Mass spectrometry demonstrated that the modification was a covalent attachment (alkylation) of TPCK at Cys55 and Cys206. Cys55 is next to the CD4 binding motif (A56W57L58) in Nef required for Nef-mediated CD4 downregulation and for AIDS development. This implies that the addition of a bulky TPCK molecule to Nef at Cys55 would impair Nef function and reduce HIV pathogenicity. As expected, Cys55 modification reduced the strength of the interaction between Nef-His and CD4 tail peptide by 50%.
Our data suggest that this Cys55-specific alkylation mechanism may be exploited to develop a new class of anti HIV drugs.
Nef proteins of primate lentiviruses, HIV-1, HIV-2 and SIV, are abundantly expressed in the early phase of HIV-1 infection and play a crucial role in the pathogenicity of HIV-1 and the development of AIDS [1–8]. One prominent piece of evidence is that HIV-1 strains isolated from some long-term survivors carried deletions or truncations of nef exclusively [9, 10]. The pathological roles of Nef in the development of AIDS have been attributed to several Nef biological activities, including downregulation of the viral primary receptor CD4  and downregulation of the cell surface expression of class-I major histocompatibility complex (MHC-I) [12, 13]. Nef also affects T cell activation and apoptosis in favor the viral replication by engaging several signaling molecules, such as Vav, Pak2, ASK1 and Src family kinases [14–18] (for reviews, see [19, 20]). Nef has no known catalytic activity; it acts essentially as a connector to link CD4, MHC-I, and possibly some other target molecules to adaptor protein (AP) complexes AP-1, AP-2 or AP-3, responsible for the endocytosis and subsequent lysosomal degradation of Nef's targets. We found that Nef-mediated CD4 downregulation is AP-2 dependent and required an ubiquitinated lysine residue K144 in HIV-1 Nef [21, 22]. The structure of HIV-1 Nef has been established by NMR and X-ray crystallography [23–25] (see  for a review). HIV-1 Nef protein consists of a conserved core domain of about 120 residues and two flexible regions - the N-terminus 68 amino acids flexible arm and a 32 amino acid loop structure (V148-L181) located in the C-terminal region. The HIV protease cleavage site C55AW57LEA  and CD4 binding motif (A56W57L58)  are located in Nef N-terminal region. Nef is myristoylated at a Gly residue (G2) in the N-terminus, which mediates the membrane association of Nef . The core domain is a α-β globular structure responsible for Nef binding to SH3 domain-containing proteins [16, 30, 31]. The loop in the C-terminal region contains the dileucine motif ExxxLL160, which interacts with adaptor protein complexes AP-1, 2, 3 [32–34].
TPCK (N-α-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-α-p-tosyl-L-lysine chloromethyl ketone) are alkylation reagents that can chemically modify side chains of specific His or Cys residues in some proteins. It is known that TPCK modifies His in the reactive center of serine protease chymotrypsin and trypsin, resulting in enzymatic inhibition (EC50 of 20 μM and 80 μM, respectively) [35, 36]. TPCK and TLCK also alkylate the sulfhydryl group of the Cys residue in several other proteins, including protein kinase C [37, 38], cAMP-dependent kinase [39, 40], HPV-18 E7  and human ETS 1 oncoprotein . Alkylation of Cys side chains makes HPV-18 E7  and human ETS 1 oncoprotein  migrate faster on SDS-PAGE.
Cells, antibodies and chemicals
SV40 T antigen-transfected human leukemic Jurkat T cells (JTAg) were cultured in RPMI medium supplemented with 10% FCS. For transient expression, plasmid DNA was transfected into the cells using Lipofectamine 2000™(Invitrogen). Anti-HIV-1 Nef rabbit serum was obtained from NIH AIDS Research and Reference Reagent Program. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), NA-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-CBZ-Phe-Ala fluoromethyl ketone (Z-FA-FMK) were purchased from Sigma (Saint Louis, MO).
HIV-1 Nef (NA7)-GFP plasmid kindly provided by Dr. J. Skowronski was subcloned into pcDNA3 to express un-tagged wt Nef (NA7). Nef (G2G3/AA) mutant was generated by PCR mutagenesis as described before . Nef (NL4-3) was PCR subcloned into pcDNA3 vector with the template of HIV-1 (NL4-3) provirion from NIH AIDS Research and Reference Reagent Program. Nef Cys-to-Ala mutants C55/A, C142/A, C206/A, C55&206/A, C55&142/A, C142&206/A and C55&C142&C206/A (Cys free) were generated by PCR mutagenesis with wt Nef (NA7) plasmid template using Multi-Quick Change Mutagenesis kit (Stratagene). For E. coli cell expression, wt Nef and Nef mutants were subcloned into pET-30a (+) vector (Novagen) at Nde I/Not I sites. All mutations generated in this study were confirmed by DNA sequencing.
Analysis of Nef modification in TPCK- or TLCK-treated JTAg cells
Analysis was performed using Nef (NA7) transfected JTAg cells unless otherwise specified. Cells were transfected with Nef plasmid DNA for 16-20 h and treated with TPCK/TLCK (10 μg/ml) for 30 min. Cells (2 × 105) were boiled in 25 μl 2 × SDS sample buffer and loaded to 11% reducing SDS-PAGE. Nef protein was detected by immunoblotting with polyclonal anti-Nef (1:10,000 dilution) at RT for 2 h or at 4°C overnight, followed by ECL anti-rabbit Ab (1:10,000) at RT for 1 h.
Nef-His protein preparation and in vitro modification
Plasmid encoding Nef-His in pET-30a (+) vector was transformed into E. coli BL21 cells. The transformed cells were grown in LB medium at 37°C for 16 h, 1: 10 diluted with fresh LB, and induced with IPTG (1 mM) for 3 hours. Four hundred ml of cells were pelleted, washed with PBS and lysed by sonication. Nef-His protein was isolated with a HisTrap column (Amersham Biosciences) or using Ni-NTA agarose beads (QIAGEN). The beads were washed three times in 20 mM Imidazole/PBS. Nef-His was eluted with 250 mM Imidazole, adjusted with PBS to the concentration of UV absorbance (A280) = 1.0, and kept at -20°C before use. For in vitro modification, freshly prepared Nef-His was incubated with TPCK (10 μg/ml) at RT for 30 min. Twenty μl of samples was resolved by SDS-PAGE. The gels were stained with Coomassie Blue or immunoblotted with anti-Nef.
Nef-His protein was in vitro modified with TPCK as described above. The completion of the modification was confirmed by SDS-PAGE. Fifty μg of the un-modified and TPCK-modified Nef-His proteins were analyzed by MS to determine the molecular weight. For trypsin-digestion, 20 μg of Nef-His was denatured in 0.1 M ammonium bicarbonate at 55°C for 30 min and then digested at 37°C with trypsin at 1:100 (w/w). The samples were subjected to mass spectrometry (MALDI-ToF) at the NYU medical school service center using MS spectrometer Micromass (Waters).
Fluorescence titration of CD4 tail peptide with HIV-1 Nef
Fluorescein-labeled CD4 tail peptide (Fluorescein-QAERMSQIKRLLSEKKT, residue 403-419) was synthesized by Sigma. Fluorescence emission was recorded with a FluoroMax-2 fluorescence spectrometer (excitation at 492 nm; emission at 516 nm). CD4 tail peptide of 1.0 μM in PBS was analyzed in a stirred cuvette at 25°C. Data were collected after 30 min incubation with Nef-His. Controls incubated with PBS did not show reduction in fluorescence. Experimental signals were expressed as the percentage of fluorescence reduction averaged from three independent measurements. The signals were plotted against total Nef concentration.
CD spectrum of Nef-His
Nef-His protein of 100 μM (~2.5 mg/ml) in PBS, pH 7.4, was subjected to CD spectrometry analysis. Far-UV CD measurement at 20°C was made on an Aviv 202 CD spectrometer (Lakewood, NJ) in the department of chemistry of NYU. Data were the average of 4-6 accumulations, using scanning wavelength of 260-195 nm, speed of 20 nm/min, bandwidth of 1 nm, and response time of 0.5 s. Data were plotted using the SigmaPlot software.
TPCK and TLCK modified HIV-1 Nef expressed in culture T cells
TPCK modified Nef at Cys55 and Cys206
TPCK modified recombinant Nef-His protein in vitro and the modification appeared to be dependent on Nef conformation
MS analysis proved that TPCK was covalently bound to Cys 55 and Cys 206 but not to His residues
TPCK alkylation at Cys55 severely impaired Nef's interaction with CD4 tail peptide
CD spectrometry data indicated a moderate Nef conformational change after TPCK alkylation
This study demonstrated that alkylation reagents, TPCK and TLCK, modify HIV-1 accessory protein Nef in live T cells and in vitro. Mutagenesis and MS analysis indicated that TPCK-modification of Nef is an alkylation reaction that resulted in the covalent bound of TPCK molecule to the side chains of Cys55 and Cys206 residues (Fig. 1, 2, 3, 4). Several lines of evidence suggest that the reaction is quite specific: (1) TPCK and TLCK have been used as specific serine protease inhibitors. The EC50 values of TPCK and TLCK alkylation on Nef are lower than that on chymotrypsin and trypsin, suggesting higher alkylation specificity than that of serine proteases. (2) Z-FA-FMK, a structurally very similar alkylation reagent, is inactive in modifying Nef (Fig. 1). (3) TPCK reacts with Cys but not with His residues, including those in the C-terminal His-tag, fully accessible to TPCK (Fig. 4). (4) TPCK appears to alkylate Cys55 more efficiently than to Cys206 (Fig.1).
The mechanism by which TPCK alkylates Cys residue is much less understood than the mechanism by which it alkylates His residues. It is well known that TPCK inhibits serine proteases by alkylating the His side chain at an enzyme' reactive center [35, 36]. This understanding has rationalized the use of TPCK in signal transduction studies. In addition, some recent reports implicated the effects of alkylation at Cys, rather than at His residues [46–48]. However, how TPCK reacts with specific His or Cys is unclear. Our study showed that in case of Nef, the accessibility of Cys residues for TPCK appeared important but not sufficient for TPCK-modification. The TPCK-modified Cys55 and Cys206 are both accessible, locating in Nef N-terminal flexible region and at the C-terminal end, respectively, whereas the none-modified C142 is buried in the Nef core . However, accessibility cannot explain why TPCK did not react with any His residues despite that there are nine His residues in Nef, of which several are accessible. They include His 40 in the N-terminal flexible region and His166/His171 in the C-terminal loop region. In addition, TPCK did not react with any His residues in the C-terminal His-tag. Probably the residues surrounding the reactive Cys or His are involved in the interaction with TPCK side chain, thus contributed to alkylation specificity.
Cys55 is next to Nef motif A56W57L58, a site important for Nef-CD4 interaction and development of AIDS . The motif is also the cleavage site for HIV protease . It is conceivable that the covalent attachment of a bulky TPCK molecule to Cys55 would interfere with Nef-CD4 interaction and some other Nef functions. Fluorescence titration data indicated that TPCK-modification indeed dramatically reduced the binding strength of Nef to a CD4 tail peptide (Fig. 5). TPCK-modification may have an additional mechanism against HIV-1 by altering Nef conformation as shown by the CD spectrum change (Fig. 6) and making it unstable as suggested by a shortened half-life of Nef in T cells also (unpublished data). Unfortunately, current cell system is not fit for testing anti HIV-1 activity due to technical difficulty. TPCK only partially (50%, maximum) alkylates wt Nef overexpressed in cultured T cells, leaving more than half of Nef without alkylation (Fig.1). A small fraction of unalkylated Nef protein is sufficient to downregulate CD4. Moreover, TPCK is toxic to T cells at high concentrations, which compromises the interpretation of an anti HIV-1 activity.
Our finding suggests that TPCK can serve as a prototype of a class of drugs that retains the Cys55 modification activity but has desired pharmacodynamic and pharmacological properties. A 3-D structure of the TPCK-bound Nef could guide the design and synthesis of new compounds. In this regard, we have developed a convenient method of generating large quantity of TPCK-bound Nef for structure studies (Fig. 3, 4). A comparison of such a 3-D structure with the existing 3-D model of TPCK bound to a His residue at the catalytic center of a serine protease  may aid the development of similar compounds that are specific for cysteine over histidine or vice versa.
Chloromethyl ketone reagents TPCK and TLCK directly react with Cys55 and Cys206 in Nef. TPCK alkylation at Cys55 dramatically weakens Nef-CD4 interaction, suggesting that TPCK-like small chemicals with better pharmacokinetics and pharmacodynamics may be developed for HIV disease intervention.
List of abbreviations
human immunodeficiency virus
SV40 large T antigen-transfected human leukemic Jurkat T cells
N-α-p-tosyl-L-phenylalanine chloromethyl ketone
N-α-p-tosyl-L-lysine chloromethyl ketone
N-CBZ-Phe-Ala fluoromethyl ketone
major histocompatibility complex class I.
We thank Tom Nubert and Chong-Feng Xu for the mass spectrometry. This work was supported by NIH grant (AI 78794) to Yong-Jiu Jin and NIH grant (AI 51214) to Xiaoping Zhang.
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