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Arnesen T.,University of Bergen | Starheim K.K.,University of Bergen | Van Damme P.,VIB | Van Damme P.,Ghent University | And 10 more authors.
Molecular and Cellular Biology | Year: 2010

The human NatA protein Nα-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G0/G1 phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Lobito A.A.,Genentech | Lobito A.A.,Catalyst Biosciences | Ramani S.R.,Genentech | Tom I.,Genentech | And 6 more authors.
Journal of Biological Chemistry | Year: 2011

Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Chao L.H.,University of California at Berkeley | Chao L.H.,Howard Hughes Medical Institute | Pellicena P.,University of California at Berkeley | Pellicena P.,Howard Hughes Medical Institute | And 9 more authors.
Nature Structural and Molecular Biology | Year: 2010

The dodecameric holoenzyme of calcium-calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca 2+ pulses to become Ca 2+ independent. A simple coincidence-detector model for Ca 2+-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca 2+-calmodulin is cooperative, with a Hill coefficient of 3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments. © 2010 Nature America, Inc. All rights reserved.

Catalyst Biosciences | Date: 2010-10-06

Biochemicals, namely, proteases for use in human therapeutics, namely, for the treatment of infectious, auto-immune, cell-proliferative, inflammatory, metabolic, cardiovascular, respiratory, neurological, and musculoskeletal disorders and diseases.

Maifeld S.V.,University of California at San Francisco | MacKinnon A.L.,University of California at San Francisco | Garrison J.L.,University of California at San Francisco | Garrison J.L.,Rockefeller University | And 7 more authors.
Chemistry and Biology | Year: 2011

Cotransins are cyclic heptadepsipeptides that bind the Sec61 translocon to inhibit cotranslational translocation of a subset of secreted and type I transmembrane proteins. The few known cotransin-sensitive substrates are all targeted to the translocon by a cleavable signal sequence, previously shown to be a critical determinant of cotransin sensitivity. By profiling two cotransin variants against a panel of secreted and transmembrane proteins, we demonstrate that cotransin side-chain differences profoundly affect substrate selectivity. Among the most sensitive substrates we identified is the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Like all type II transmembrane proteins, TNF-α is targeted to the translocon by its membrane-spanning domain, indicating that a cleavable signal sequence is not strictly required for cotransin sensitivity. Our results thus reveal an unanticipated breadth of translocon substrates whose expression is inhibited by Sec61 modulators. © 2011 Elsevier Ltd. All rights reserved.

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