San Francisco, 9-13 December 2000. Report by Mervyn J. Monteiro, University of
Maryland Biotechnology Institute

The 40th ASCB meeting began its grand kick-off on Saturday December 9 with
an opening symposium by four Nobel Laureates, J. Michael Bishop, Joseph Goldstein,
Michael Brown, and Harold Varmus. Bishop summarized his discovery of proto-oncogenes,
and went on to describe recent studies from his laboratory involving one such
proto-oncogene, MYC, and its roles in controlling cellular proliferation and
tumorigenesis. He described the phenotypes of transgenic mice that were engineered
to have tetracycline-inducible expression of the MYC gene in hematopoietic cells.
Bishop’s group found that overexpression of MYC caused malignant T cell lymphomas
and acute myeloid leukemias. Remarkably, these cancers regressed when the mice
were treated for two weeks with doxycycline (which turned off expression of
the tetracycline-regulated MYC transgene). He showed that rapid proliferative
arrest, differentiation and apoptosis of tumor cells, and resumption of normal
host hematopoiesis accompanied this reversal of lymphomas in the transgenic
mice. Bishop mentioned that other investigators have also successfully reversed
tumors by selectively turning-off other proto-oncogenes, and alluded to the
possibility that some tumors and cancers could be reversed if expression of
proto-oncogenes could be selectively controlled in humans.

Reference:

Mol Cell 4:199-207.

Goldstein and Brown, in a duet presentation, described Regulated Intramembrane
Proteolysis (RIP), a process by which membrane spanning proteins are cleaved
at sites within their hydrophobic transmembrane domains (TMD). RIP can be subdivided
into two classes. Type 1 transmembrane proteins are orientated with their NH2-termini
facing the lumen of the ER and/or Golgi and their COOH termini in the cytosol,
and typified by transmembrane proteins such as APP, Notch and IRE1 which are
believed to be cleaved by presenilins. Type 2 transmembrane proteins, which
have their NH2-termini facing the cytosol, include the transcriptional factors
SREBP and ATF6. An understanding of how RIP is controlled should provide useful
information regarding proteolytic cleavage of transmembrane proteins, for example,
how APP is cleaved to generate Aβ, the primary component of plaques in Alzheimer’s
disease (AD), and whether such an activity is aberrant in AD.

Goldstein and Brown described how RIP regulates the activities of SREBP and
ATF6, transcriptional factors that regulate cholesterol biosynthesis and the
unfolded protein response, respectively. SREBP is a long protein of ~1150 amino
acids containing a basic helix-loop-helix-leucine (bHLH) domain at its NH2-terminus
and a regulatory domain at its COOH-terminus. These two domains are separated
from each other by two TMDs, which are themselves separated by a short 30 amino
acid loop segment that protrudes into the lumen. When cholesterol levels in
cells are low, SREBP is cleaved such that the N-terminal bHLH is released and
translocates into the nucleus, where it activates genes involved in cholesterol
biogenesis. This cleavage of SREBP actually occurs in a step-wise fashion: the
first cut is made at site 1 (RSVL(S) within the loop segment spanning the two
TMDs by a protease called Site 1 Protease (S1P), while the second cut, at site
2 (ILL(C), which is located three amino acids downstream in TMD1, is made by
Site 2 Protease (S2P). Goldstein’s group has characterized the S1P and S2P activities,
and has generated cell lines that lack expression of one or both of the proteases.
These mutant cell lines provided valuable insight into how cholesterol regulates
SREBP cleavage. When cells are grown in conditions that promote high cholesterol
levels, SREBP binds to WD repeats located in the C-terminus of SCAP (an 8 TMD-containing
protein), and this protein-protein interaction causes the SREBP/SCAP complex
to be trapped in the endoplasmic reticulum (ER). For SREBP to be cleaved, it
must exit the ER and be transported to post-ER compartments, such as the Golgi,
where the active subtilisin like serine S1P is located. Interestingly, although
S2P, a zinc-metalloprotease, is not regulated by cholesterol, it cannot cleave
SREBP if site 1 cleavage has not occurred. Thus high levels of cholesterol prevent
SREBP processing, which prevents its N-terminal bHLH domain from activating
further cholesterol biosynthesis, thereby ensuring negative feedback control.
However, when cells are grown in conditions in which cholesterol is limiting,
the SCAP/SREPB complex is translocated to the Golgi, where S1P cleaves SREPB.
This cleavage is believed to cause partial unwinding of the TMD1 α-helix, which
makes site 2 (Cys(Leu) accessible for cleavage by S2P. This second cleavage
results in release of the bHLH transcription factor, which activates transcription
of enzymes involved in cholesterol and fatty acid synthesis.

Likewise, the cleavage of ATF6, which is involved in the unfolded protein response,
is also regulated by a two-step process involving S1P and S2P proteases. ATF6
contains an NH2-terminal basic zipper domain followed by one TMD spanning-segment
and a “sensor” domain in its C-terminus that is located in the lumen of the
ER. Under conditions of ER stress (for example, when cells are treated with
tunicamycin which blocks glycosylation and causes protein misfolding) ATF6 is
cleaved at site 1 (RHL(L) located downstream of the TMD. This cleavage leads
to partial unwinding of the α-helical TMD and allows subsequent cleavage at
an upstream site located within the TDM segment. Upon cleavage at both site
1 and 2, the NH2-terminal zipper domain of AT6 translocates into the nucleus
where it activates genes involved in the unfolded protein response, such as,
Bip/GRP78, GRP94 and calreticulin. Interestingly, both ATF6 and SREBP contain
two Asn Pro residues, nearby or adjacent to one another within their cleaved
TDM segments, both of which are essential for the presumed partial unwinding
and subsequent cleavage of site 2. It will be interesting to determine if similar
or different mechanisms are involved in APP, Notch and IRE1 cleavage.

 References:

Cell 99:703-12;

Cell 102:315-323;

PNAS 97:5123-28;

Mol. Biol. Cell 10:3787-99

Cell 100: 391-98.

Varmus talked about how the explosion of biomedical information from various
genome sequencing projects and technical advances in microarray screens and
proteomics has necessitated that the information be properly archived and readily
accessible to the entire scientific community worldwide. He pointed out that
NIH was actively engaged in this endeavor as part of the Biomedical Information
and Technology Initiative. He also stressed his belief that life-science publications
should be freely accessible to all and that PubMed Central (http://www.pubmedcentral.nih.gov/),
an e-biomed system which he helped establish, is rapidly growing as more journals
are joining in this initiative. He stressed that both peer-reviewed as well
as non-peer reviewed articles (screened by independent organizations) will be
accessible through PubMed Central. He said that to alleviate some of the expenses
associated with publishing, costs would be shifted to authors, rather than readers,
as the readers are most often the authors!


References: Felsher DW, Bishop JM. Reversible tumorigenesis by MYC in hematopoietic lineages.Mol Cell 1999 Aug;4(2):199-207. Abstract

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References

Paper Citations

  1. . Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell. 1999 Aug;4(2):199-207. PubMed.

External Citations

  1. Mol. Biol. Cell 10:3787-99

Further Reading

Papers

  1. . Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell. 1999 Aug;4(2):199-207. PubMed.

Primary Papers

  1. . Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell. 1999 Aug;4(2):199-207. PubMed.