Limits...
The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients.

Van Duyne R, Guendel I, Kehn-Hall K, Easley R, Klase Z, Liu C, Young M, Kashanchi F - AIDS Res Ther (2010)

Bottom Line: We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs).We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells.Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.

View Article: PubMed Central - HTML - PubMed

Affiliation: George Mason University, Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, Manassas, VA 20110, USA. bcmfxk@gwumc.edu.

ABSTRACT

Background: The search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. The comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. In particular, the analysis of HIV-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. The utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to HIV-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum.

Results: Here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs). Here we have identified unique serum proteins that are differentially expressed in LTNP HIV-1 patients and may contribute to the ability of these patients to combat HIV-1 infection in the absence of HAART. We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells.

Conclusions: Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.

No MeSH data available.


Related in: MedlinePlus

Western blot confirmation of MALDI-TOF identified serum proteins. A) Western blots were performed against pooled low (L) and high (H) abundance protein fractions for negative (lanes 2, 3), LTNP (lanes 4, 5), and HAART (lanes 6, 7) patients. Antibodies specific to cdk4, p16INK4A, PCTAIRE, HP1α, and HP1γ were used. B) Western blots were performed against individual patient samples 1-6, low and high abundant LTNPs. Antibodies specific to p16INK4A and cdk4 were used. 293T, the pooled low abundance LTNP samples "A," and the pooled high abundance LTNP samples "D" were used as controls. C) Immunoprecipitation of p16INK4A from the individual low abundant LTNP patient samples, followed by a western blot against p16INK4A. HeLa whole cell extract and the pooled low abundance LTNP sample "A" were used as controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908552&req=5

Figure 3: Western blot confirmation of MALDI-TOF identified serum proteins. A) Western blots were performed against pooled low (L) and high (H) abundance protein fractions for negative (lanes 2, 3), LTNP (lanes 4, 5), and HAART (lanes 6, 7) patients. Antibodies specific to cdk4, p16INK4A, PCTAIRE, HP1α, and HP1γ were used. B) Western blots were performed against individual patient samples 1-6, low and high abundant LTNPs. Antibodies specific to p16INK4A and cdk4 were used. 293T, the pooled low abundance LTNP samples "A," and the pooled high abundance LTNP samples "D" were used as controls. C) Immunoprecipitation of p16INK4A from the individual low abundant LTNP patient samples, followed by a western blot against p16INK4A. HeLa whole cell extract and the pooled low abundance LTNP sample "A" were used as controls.

Mentions: In order to further confirm the presence of these proteins in the serum as identified by mass spectrometry, we performed western blots on the same low and high abundant pooled fractions (Figure 3A). P16INK4A is present in both the low and high abundant fractions of the pooled LTNPs (lanes 3, 4) and is also observed in the high abundance fraction of uninfected patients (Figure 3A, lane 2). Interestingly, this protein is not present in HAART patient samples at all (Figure 3A, lane 5, 6). The presence of this protein in serum may be specific to individuals that confer resistance to chronic HIV-1 infection. As p16INK4A is an inhibitor of cell cycle kinases, in particular cdk4 and cdk6, the levels of cdk4 in the serum samples was assayed and was shown to be ubiquitously expressed across all low and high abundance serum samples (Figure 3A, third panel from top). Indeed, levels of cdk6 were not detectable in any of the patient serum samples as compared to a 293T whole cell extract positive control (data not shown). This implies that the presence of cdk4 in the serum is not dependent on the presence or absence of p16INK4A and likewise, p16INK4A does not affect the expression levels of cdk4 amongst the patient serum samples. The HP1 binding protein was initially identified in the 1D/mass spectrometry analysis in the LTNP low abundance fraction, therefore the serum levels of both HP1α and HP1γ subunits were assayed (Figure 3A). The family of heterochromatin-associated proteins exist as three distinct isoforms, α, β, and γ and all act as regulators of heterochromatin-mediated transcriptional silencing [47]. HP1α has been shown to directly interact with DNA methyltransferases and histone methyltransferases to mediate transcriptional silencing [48,49] and HP1γ, in particular, interacts with the histone methyltransferase Suv39H1 to initiate a chromatin-mediated repressive state of the HIV-1 integrated virus [50]. HP1α was shown to be present in the low abundance fractions of all of the patient phenotypes whereas HP1γ was shown to be present in both the low and high abundant fractions across all patient types (Figure 3A). HP1γ is observed in lower amounts in both the Negative and HAART high abundance fractions and all serum samples indicate the presence of a post-translational modification (i.e. a doublet band) as compared to the 293T whole cell extract positive control. This indicates that the HP1γ found in serum exists in both a modified and unmodified form. Interestingly, PCTAIRE was present in the highest abundance in the uninfected (Negative), high abundance fraction (Figure 3A, lane 2), however low levels were also seen in both LTNP and HAART high abundance fractions (Figure 3A, lane 4, 6). PCTAIRE was identified initially by mass spectrometry in the high abundance LTNP sample and can be seen in the high abundance fractions of all three of the patient types biochemically, however it is present in lower amounts in the HIV-1 infected patients, indicating that this kinase may be differentially expressed upon infection though not necessarily a unique identifier for infection. P16INK4A is the only protein identified from mass spectrometric analysis and confirmed biochemically that is specific for the low abundance LTNP serum samples; although the protein is also identified in the uninfected and the high abundance LTNP fractions. These results are also interesting due to the involvement of p16INK4A in alterations of cell cycle control, additionally, mutations in p16INK4A are found in various cancers including pancreatic, lymphomas, and sarcomas, contributing to cancer progression [45]. These findings also indicate a difference in composition of serum proteins present in HIV-1 infected individuals undergoing HAART treatment versus those that are naturally non-progressing.


The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients.

Van Duyne R, Guendel I, Kehn-Hall K, Easley R, Klase Z, Liu C, Young M, Kashanchi F - AIDS Res Ther (2010)

Western blot confirmation of MALDI-TOF identified serum proteins. A) Western blots were performed against pooled low (L) and high (H) abundance protein fractions for negative (lanes 2, 3), LTNP (lanes 4, 5), and HAART (lanes 6, 7) patients. Antibodies specific to cdk4, p16INK4A, PCTAIRE, HP1α, and HP1γ were used. B) Western blots were performed against individual patient samples 1-6, low and high abundant LTNPs. Antibodies specific to p16INK4A and cdk4 were used. 293T, the pooled low abundance LTNP samples "A," and the pooled high abundance LTNP samples "D" were used as controls. C) Immunoprecipitation of p16INK4A from the individual low abundant LTNP patient samples, followed by a western blot against p16INK4A. HeLa whole cell extract and the pooled low abundance LTNP sample "A" were used as controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908552&req=5

Figure 3: Western blot confirmation of MALDI-TOF identified serum proteins. A) Western blots were performed against pooled low (L) and high (H) abundance protein fractions for negative (lanes 2, 3), LTNP (lanes 4, 5), and HAART (lanes 6, 7) patients. Antibodies specific to cdk4, p16INK4A, PCTAIRE, HP1α, and HP1γ were used. B) Western blots were performed against individual patient samples 1-6, low and high abundant LTNPs. Antibodies specific to p16INK4A and cdk4 were used. 293T, the pooled low abundance LTNP samples "A," and the pooled high abundance LTNP samples "D" were used as controls. C) Immunoprecipitation of p16INK4A from the individual low abundant LTNP patient samples, followed by a western blot against p16INK4A. HeLa whole cell extract and the pooled low abundance LTNP sample "A" were used as controls.
Mentions: In order to further confirm the presence of these proteins in the serum as identified by mass spectrometry, we performed western blots on the same low and high abundant pooled fractions (Figure 3A). P16INK4A is present in both the low and high abundant fractions of the pooled LTNPs (lanes 3, 4) and is also observed in the high abundance fraction of uninfected patients (Figure 3A, lane 2). Interestingly, this protein is not present in HAART patient samples at all (Figure 3A, lane 5, 6). The presence of this protein in serum may be specific to individuals that confer resistance to chronic HIV-1 infection. As p16INK4A is an inhibitor of cell cycle kinases, in particular cdk4 and cdk6, the levels of cdk4 in the serum samples was assayed and was shown to be ubiquitously expressed across all low and high abundance serum samples (Figure 3A, third panel from top). Indeed, levels of cdk6 were not detectable in any of the patient serum samples as compared to a 293T whole cell extract positive control (data not shown). This implies that the presence of cdk4 in the serum is not dependent on the presence or absence of p16INK4A and likewise, p16INK4A does not affect the expression levels of cdk4 amongst the patient serum samples. The HP1 binding protein was initially identified in the 1D/mass spectrometry analysis in the LTNP low abundance fraction, therefore the serum levels of both HP1α and HP1γ subunits were assayed (Figure 3A). The family of heterochromatin-associated proteins exist as three distinct isoforms, α, β, and γ and all act as regulators of heterochromatin-mediated transcriptional silencing [47]. HP1α has been shown to directly interact with DNA methyltransferases and histone methyltransferases to mediate transcriptional silencing [48,49] and HP1γ, in particular, interacts with the histone methyltransferase Suv39H1 to initiate a chromatin-mediated repressive state of the HIV-1 integrated virus [50]. HP1α was shown to be present in the low abundance fractions of all of the patient phenotypes whereas HP1γ was shown to be present in both the low and high abundant fractions across all patient types (Figure 3A). HP1γ is observed in lower amounts in both the Negative and HAART high abundance fractions and all serum samples indicate the presence of a post-translational modification (i.e. a doublet band) as compared to the 293T whole cell extract positive control. This indicates that the HP1γ found in serum exists in both a modified and unmodified form. Interestingly, PCTAIRE was present in the highest abundance in the uninfected (Negative), high abundance fraction (Figure 3A, lane 2), however low levels were also seen in both LTNP and HAART high abundance fractions (Figure 3A, lane 4, 6). PCTAIRE was identified initially by mass spectrometry in the high abundance LTNP sample and can be seen in the high abundance fractions of all three of the patient types biochemically, however it is present in lower amounts in the HIV-1 infected patients, indicating that this kinase may be differentially expressed upon infection though not necessarily a unique identifier for infection. P16INK4A is the only protein identified from mass spectrometric analysis and confirmed biochemically that is specific for the low abundance LTNP serum samples; although the protein is also identified in the uninfected and the high abundance LTNP fractions. These results are also interesting due to the involvement of p16INK4A in alterations of cell cycle control, additionally, mutations in p16INK4A are found in various cancers including pancreatic, lymphomas, and sarcomas, contributing to cancer progression [45]. These findings also indicate a difference in composition of serum proteins present in HIV-1 infected individuals undergoing HAART treatment versus those that are naturally non-progressing.

Bottom Line: We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs).We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells.Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.

View Article: PubMed Central - HTML - PubMed

Affiliation: George Mason University, Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, Manassas, VA 20110, USA. bcmfxk@gwumc.edu.

ABSTRACT

Background: The search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. The comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. In particular, the analysis of HIV-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. The utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to HIV-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum.

Results: Here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs). Here we have identified unique serum proteins that are differentially expressed in LTNP HIV-1 patients and may contribute to the ability of these patients to combat HIV-1 infection in the absence of HAART. We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells.

Conclusions: Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.

No MeSH data available.


Related in: MedlinePlus