Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.

BACKGROUND.Early detection of hepatocellular carcinoma (HCC), one of the most common and deadly tumors worldwide, still is difficult due to the lack of adequate biomarkers that show high sensitivity and specificity. The authors recently demonstrated that squamous cell carcinoma antigen (SCCA) variants were overexpressed remarkably in all surgically resected HCCs. METHODS.For the current study, the authors assessed the presence of SCCA, as a free form and complexed with immunoglobulins, in serum from patients with HCC, cirrhosis, and chronic hepatitis and from healthy control participants and compared SCCA measurement with the measurement of ␣-fetoprotein (AFP) levels. RESULTS.Circulating immune complexes (ICs) composed by SCCA and immunoglobulin M (IgM) IC (SCCA-IgM IC) were undetectable (Ͻ 120 arbitrary units[AU]/mL) in serum from a healthy control population (0 of 73 controls); however, 35 of 50 patients with HCC (70%) were reactive for SCCA-IgM IC independent of etiology (mean Ϯ standard deviation [SD], 2568.5 Ϯ 6797.3 AU/mL). No correlation was found with AFP levels, which were elevated significantly in only 21 of 50 patients with HCC (42%). By using an AFP cut-off value of 20 ng/mL, 96% of patients with HCC were positive for at least 1 marker. Among cirrhotic patients, the presence of circulating SCCA-IgM IC was displayed in 13 of 50 patients (26%), but at lower levels compared with the patients who had HCC (mean Ϯ SD, 147.5 Ϯ 348.3 AU/mL; P Ͻ 0.01; Student t test), whereas 9 of 50 patients with chronic hepatitis (18%) were reactive (mean Ϯ SD, 39.5 Ϯ 89.7 AU/mL). No significant presence of free SCCA, free anti-SCCA variants IgG or IgM, or SCCA-IgG IC was found. CONCLUSIONS.The study results indicated that SCCA-IgM ICs represent novel serologic biomarkers, which, alone or in combination with AFP, can increase the sensitivity for diagnosing HCC significantly.

Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre‐Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N‐ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER‐Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide‐linked to Cys214. We suggest that thiol‐mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.

Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (scorep1) was associated to lower MIB-1 labelling index, compared to cases with SCCA score X2 (mean7s.d.: 2%72.4 vs 7.5%710.3, Po0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G 351 to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours. Hepatocellular carcinoma (HCC) is one of the most important sanitary problems over the world for its high prevalence and for its poor prognosis. More than 250 000 new cases per year are diagnosed and mean 5-year survival is lower than 5% (El-Serag and Mason, 1999). Several factors have been involved in the development of liver cancer, some of them differing in various geographic areas and explaining, at least in part, distinct geographical incidence (Bruix et al, 2001). The most powerful risk factor is the presence of liver cirrhosis. Among cirrhotic Caucasian patients, chronic viral infection by HBV and HCV are the most biologically relevant causes leading to this clinical condition (Benvegnù et al, 1994). However, pathogenetic mechanisms of neoplastic transformation are still unclear; several growth factors and tumour suppressor genes (Collier et al, 1993; Naka et al, 1998; Abou-Shady et al, 1999) have been involved but insufficient data have been generated to date. cDNA microarray analyses for gene expression profiling and potential identification of target genes for diagnostic or therapeutic purposes are ongoing (Shirota et al, 2001;Takeo et al, 2001;Xu et al, 2001).Squamous cell carcinoma antigen is a serine protease inhibitor physiologically found in the spinous and granular layers of normal squamous epithelium, but typically expressed by neoplastic cells of epithelial origin (Kato, 1996). Recent studies indicate that both SCCA1 and SCCA2, the two isoforms so far identified (Schneider et al, 1995), protect neoplastic cells from apoptotic death induced by several k...

The stability of oligomeric human tumour necrasis factor alpha (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37 degrees C) at 0.12 nM, whereas no apparent dissociation was detected at 4 nM. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nM by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.