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P A Bresnahan

Publications and source records attributed to P A Bresnahan.

7 recordsLinked to original sources
PubMed1999

Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4.

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor.

Human immunodeficiency virus 1 (HIV-1) Nef downregulates surface expression of CD4, an integral component of the functional HIV receptor complex, through accelerated endocytosis of surface receptors and diminished transport of CD4 from the Golgi network to the plasma membrane. HIV-1 Nef also diminishes surface expression of major histocompatibility complex (MHC) class I antigens. In the case of HIV-2 and simian immunodeficiency virus 1 (SIV-1) Nef, aminoterminal tyrosine-based motifs mediate the binding of Nef to the AP-1 and AP-2 adaptors and this interaction appears to be required for CD4 downregulation. As these tyrosine motifs are not present in the HIV-1 Nef protein, the molecular basis for the presumed interaction of Nef with components of the endocytic machinery is unknown. Here, we identify a highly conserved dileucine motif in HIV-1 Nef that is required for downregulation of CD4. This motif acts as an internalization signal in the context of a CD8-Nef chimera or in a fusion of the interleukin-2 receptor alpha with an 11-amino-acid region from Nef containing the dileucine motif. Finally, HIV-1 Nef binds to the AP-1 adaptor, both in vitro and in vivo, in a dileucine-dependent manner. We conclude that this conserved dileucine motif in HIV-1 Nef serves as a key interface for interaction with components of the host protein trafficking machinery. Our findings also reveal an evolutionary difference between HIV-1 and HIV-2/SIV in which the Nef proteins utilize structurally distinct motifs for binding cellular adaptors.

Adaptor Protein Complex alpha Subunits↗
PubMed1997

Localization of class I histocompatibility molecule assembly by subfractionation of the early secretory pathway.

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with beta 2-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 degrees C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected suggesting empty molecules recycle to acquire peptide.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.

Amino Acid Sequence↗
PubMed1990

Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo.

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta-nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1982

Evaluation of density gradient separation methods.

A new method for evaluating the effectiveness of isopycnic separation processes is developed and explored. This technique is designed to measure the ability of a separation technique to attain the goals of a sorting process. The performance of a separation process is expressed in terms of an inconsistency number, I, whose value ranges from zero to unity. Processes which function perfectly are characterized by an I value of zero. Separation processes which merely subdivide a sample are characterized by an I value of unity. The validity of using I to measure the performance of separation processes was established by demonstrating that alterations in the performance of a well characterized separation technique were fully reflected by changes in the I value for the process. The capacity of four different techniques for ordering red cells with respect to their density was then shown to lie in a definite hierarchy. This hierarchy can be expressed as: Murphy technique (I = 0.21) greater than albumin density gradient (I = 0.43) greater Stractan density gradient (I = 0.53) greater than Percoll density gradient (I = 0.73). The performance was found to be dependent on the exact operating conditions employed and the specific fraction of red cells isolated.

Cell Separation↗